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EP2166069B2 - Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques - Google Patents
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EP2166069B2 - Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques - Google Patents

Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques Download PDF

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EP2166069B2
EP2166069B2 EP09176083.5A EP09176083A EP2166069B2 EP 2166069 B2 EP2166069 B2 EP 2166069B2 EP 09176083 A EP09176083 A EP 09176083A EP 2166069 B2 EP2166069 B2 EP 2166069B2
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seq
acid
desaturase
fatty acids
nucleic acid
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EP2166069A2 (fr
EP2166069B1 (fr
EP2166069A3 (fr
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Thorsten Zank
Jörg BAUER
Petra Cirpus
Amine Abbadi
Ernst Heinz
Xiao Qiu
Patricia Vrinten
Petra Sperling
Frederic Domergue
Astrid Meyer
Jelena Kirsch
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BASF Plant Science GmbH
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BASF Plant Science GmbH
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • A—HUMAN NECESSITIES
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    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings or cooking oils
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
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    • A—HUMAN NECESSITIES
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    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
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    • A23D9/02—Other edible oils or fats, e.g. shortenings or cooking oils characterised by the production or working-up
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6431—Linoleic acids [18:2[n-6]]
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    • C12P7/6445—Glycerides
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Definitions

  • the present invention relates to a method for producing polyunsaturated fatty acids in an organism by introducing nucleic acids into the organism which code for polypeptides with ⁇ 5-elongase activity.
  • nucleic acid sequences can advantageously be expressed in the organism together with other nucleic acid sequences which code for polypeptides of the biosynthesis of fatty acid or lipid metabolism.
  • Nucleic acid sequences which code for a ⁇ 6-desaturase, a ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 12-desaturase and/or ⁇ 6-elongase activity are particularly advantageous.
  • the invention further relates to a process for producing oils and/or triacylglycerides with an increased content of long-chain polyunsaturated fatty acids.
  • the present invention also relates, in a preferred embodiment, to a process for producing unsaturated ⁇ 3 fatty acids and a process for producing triglycerides with an increased content of unsaturated fatty acids, especially ⁇ 3 fatty acids with more than three double bonds.
  • the invention relates to the production of a transgenic non-human organism, preferably a transgenic plant or a transgenic microorganism, with an increased content of unsaturated ⁇ 3 fatty acids, oils or lipids with ⁇ 3 double bonds due to the expression of the elongases and desaturases used in the method according to the invention, advantageously in conjunction with ⁇ 3-desaturases, for example an ⁇ 3-desaturase from fungi of the family Pythiaceae such as the genus Phytophtora, for example the genus and species Phytophtora infestans, or an ⁇ 3-desaturase from algae such as the family Prasinophyceae, for example the genus Ostreococcus, especially the genus and species Ostreococcus tauri, or diatoms such as the genus Thalassiosira specifically the genus and species Thalassiosira pseudonana.
  • a further part of the invention relates to oils, lipids and/or fatty acids produced by the process according to the invention and their use.
  • the invention also relates to unsaturated fatty acids and triglycerides with an increased content of unsaturated fatty acids and their use.
  • Fatty acids and triacylglycerides have a variety of applications in the food industry, animal nutrition, cosmetics and pharmaceuticals. Depending on whether they are free saturated and unsaturated fatty acids or triacylglycerides with an increased saturated or unsaturated fatty acid content, they are suitable for a wide variety of applications.
  • Polyunsaturated fatty acids such as linoleic and linolenic acid are essential for mammals because they cannot produce them themselves. Therefore, polyunsaturated ⁇ 3 fatty acids and ⁇ 6 fatty acids are an important component of animal and human nutrition.
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • polyunsaturated ⁇ 3 fatty acids which are primarily found in fish oils, to the food.
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • the unsaturated fatty acid DHA is said to have a positive effect on the development and maintenance of brain functions.
  • polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or LCPUFAs ( poly unsaturated fatty acids, PUFA , polyunsaturated fatty acids; long chain poly unsaturated fatty acids, LCPUFA, long-chain polyunsaturated fatty acids) .
  • the free fatty acids are advantageously produced by saponification.
  • Common natural sources of these fatty acids are fish such as herring, salmon, sardines, yellow perch, eel, carp, trout, halibut, mackerel, zander or tuna or algae.
  • oils with saturated or unsaturated fatty acids are preferred.
  • lipids with unsaturated fatty acids especially polyunsaturated fatty acids
  • the polyunsaturated ⁇ 3 fatty acids are said to have a positive effect on the cholesterol level in the blood and thus on the possibility of preventing heart disease. Adding these ⁇ 3 fatty acids to your diet can significantly reduce the risk of heart disease, stroke or high blood pressure.
  • Inflammatory processes, especially chronic inflammatory processes in the context of immunological diseases such as rheumatoid arthritis can also be positively influenced by ⁇ 3 fatty acids. They are therefore added to foods specifically for diet foods or are used in medications.
  • ⁇ 6 fatty acids such as arachidonic acid tend to have a negative effect on these rheumatic diseases due to our usual food composition.
  • ⁇ 3 and ⁇ 6 fatty acids are precursors of tissue hormones, the so-called eicosanoids such as the prostaglandins, which are derived from dihomo- ⁇ -linolenic acid, arachidonic acid and eicosapentaenoic acid, and the thromoxanes and leukotrienes, which are derived from arachidonic acid and eicosapentaenoic acid.
  • Eicosanoids (so-called PG 2 series), which are formed from ⁇ 6 fatty acids, usually promote inflammatory reactions, while eicosanoids (so-called PG 3 series) made from ⁇ 3 fatty acids have little or no pro-inflammatory effect.
  • membrane-bound desaturases are characterized by introducing them into a suitable organism, which is then examined for enzyme activity using educt and product analysis.
  • ⁇ 6-desaturases are in WO 93/06712 , US 5,614,393 , WO 96/21022 , WO 00/21557 and WO 99/27111 described and also the application for production in transgenic organisms described as in WO 98/46763 WO 98/46764 , WO 9846765 .
  • the expression of various desaturases such as in WO 99/64616 or WO 98/46776 and formation of polyunsaturated fatty acids described and claimed.
  • microorganisms for the production of PUFAs are microorganisms such as microalgae such as Phaeodactylum tricornutum, Porphiridium species, Thraustochytria species, Schizochytria species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor and/or mosses such as Physcomitrella, Ceratodon and Marchantia ( R. Vazhappilly & F. Chen (1998) Botanica Marina 41: 553-558 ; K. Totani & K. Oba (1987) Lipids 22: 1060-1062 ; M.
  • microalgae such as Phaeodactylum tricornutum, Porphiridium species, Thraustochytria species, Schizochytria species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fung
  • EPA and DHA are produced in marine bacteria such as Vibrio sp. or Shewanella sp. by the polyketide pathway ( Yu, R. et al. Lipids 35:1061-1064, 2000 ; Takeyama, H. et al. Microbiology 143:2725-2731, 1997 ).
  • the polyunsaturated fatty acids can be divided into two large classes, ⁇ 6 or ⁇ 3 fatty acids, which have different metabolic and functional activities ( Figure 1 ).
  • the fatty acid linoleic acid (C 18:2 ⁇ 9,12 ) acts as the starting product for the ⁇ 6 metabolic pathway, while the ⁇ 3 pathway occurs via linolenic acid (C 18:3 ⁇ 9,12,15 ).
  • Linolenic acid is formed by the activity of a ⁇ 3-desaturase ( Tocher et al. 1998, Prog. Lipid Res. 37, 73-117 ; Domergue et al. 2002, Eur. J. Biochem. 269, 4105-4113 ).
  • ⁇ 3 fatty acids shows the therapeutic effect in the treatment of cardiovascular diseases as described above ( Shimikawa 2001, World Rev. Nutr. Diet. 88, 100-108 ), inflammation ( Calder 2002, Proc. Nutr. Soc. 61, 345-358 ) and arthridis ( Cleland and James 2000, J. Rheumatol. 27, 2305-2307 ).
  • the extension of fatty acids by 2 or 4 carbon atoms by elongases is of crucial importance for the production of C 20 or C 22 PUFAs. This process takes place over 4 stages.
  • the first step is the condensation of malonyl-CoA onto the fatty acid acyl-CoA by ketoacyl-CoA synthase (KCS, referred to as elongase in the following text).
  • KCS ketoacyl-CoA synthase
  • KCR ketoacyl-CoA reductase
  • dehydratase dehydration step
  • enoyl-CoA reductase enoyl-CoA reductase
  • Drexler et al 2003, Journal of Plant Physiology 160(7):779-802 describes that using ⁇ 5-elongases ARA (C 20:4 ) or EPA (C 20:5 ) can be extended by a C2 body. Reference is made to a mouse elongase described by Moon et al 2001.
  • Higher plants contain polyunsaturated fatty acids such as linoleic acid (C 18:2 ) and linolenic acid (C 18:3 ).
  • ARA, EPA and DHA do not occur at all or only in trace amounts in the seed oil of higher plants ( E. Ucciani: Wunsch Dictionnaire des Huiles Vegetales. Technique & Documentation - Lavoisier, 1995. ISBN: 2-7430-0009-0 ).
  • LCPUFAs in higher plants, preferably in oilseeds such as rapeseed, linseed, sunflower and soy, as in this way large quantities of high-quality LCPUFAs can be obtained cost-effectively for the food industry, animal nutrition and for pharmaceutical purposes.
  • genes encoding enzymes for the biosynthesis of LCPUFAs must advantageously be introduced and expressed in oilseeds using genetic engineering methods. These are genes that code for, for example, ⁇ 6-desaturases, ⁇ 6-elongases, ⁇ 5-desaturases or ⁇ 4-desaturases. These genes can be advantageously isolated from microorganisms and lower plants that produce LCPUFAs and incorporate them into the membranes or triacylglycerides. ⁇ 6-desaturase genes have already been isolated from the moss Physcomitrella patens and ⁇ 6-elongase genes from P. patens and the nematode C. elegans.
  • the first transgenic plants that contain and express genes encoding enzymes of LCPUFA biosynthesis and produce LCPUFAs were, for example, in DE 102 19 203 (Process for producing polyunsaturated fatty acids in plants) was described for the first time. However, these plants produce LCPUFAs in quantities that need to be further optimized to process the oils contained in the plants.
  • R 2 means hydrogen, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, lyso-phosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol or saturated or unsaturated C 2 -C 24 - alkylcarbonyl.
  • alkyl radicals which may be substituted or unsubstituted, saturated or unsaturated C 2 -C 24 alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl, n- Octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n- Octadecylcarbonyl, n-nonadecylcarbonyl, n-
  • C 10 -C 22 alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl.
  • n-octadecylcarbonyl n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl containing one or more double bonds are preferred.
  • saturated and/or unsaturated C 10 -C 22 alkylcarbonyl radicals such as C 10 alkylcarbonyl, C 11 alkylcarbonyl, C 12 alkylcarbonyl, C 13 alkylcarbonyl, C 14 alkylcarbonyl, C 16 alkylcarbonyl, C 18 alkylcarbonyl, C 20 alkylcarbonyl or C 22 alkylcarbonyl radicals containing one or more double bonds.
  • Saturated or unsaturated C 16 -C 22 alkylcarbonyl radicals such as C 16 alkylcarbonyl, C 18 alkylcarbonyl, C 20 alkylcarbonyl or C 22 alkylcarbonyl radicals which contain one or more double bonds are very particularly preferred.
  • These advantageous residues can contain two, three, four, five or six double bonds.
  • the particularly advantageous residues with 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, particularly preferably five or six double bonds. All residues mentioned are derived from the corresponding fatty acids.
  • R 3 in the general formula II means hydrogen, saturated or unsaturated C 2 -C 24 alkylcarbonyl.
  • alkyl radicals which may be substituted or unsubstituted, saturated or unsaturated C 2 -C 24 alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl, n- Octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n- Octadecylcarbonyl, n-nonadecylcarbonyl, n-
  • C 10 -C 22 alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl.
  • n-octadecylcarbonyl n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl containing one or more double bonds are preferred.
  • C 10 alkylcarbonyl radicals such as C 10 alkylcarbonyl, C 11 alkylcarbonyl, C 12 alkylcarbonyl, C 13 alkylcarbonyl, C 14 alkylcarbonyl, C 16 alkylcarbonyl are particularly preferred -, C 18 alkylcarbonyl, C 20 alkylcarbonyl or C 22 alkylcarbonyl radicals containing one or more double bonds.
  • Saturated or unsaturated C 16 -C 22 alkylcarbonyl radicals such as C 16 alkylcarbonyl, C 18 alkylcarbonyl, C 20 alkylcarbonyl or C 22 alkylcarbonyl radicals which contain one or more double bonds are very particularly preferred.
  • These advantageous residues can contain two, three, four, five or six double bonds.
  • the particularly advantageous residues with 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, particularly preferably five or six double bonds. All residues mentioned are derived from the corresponding fatty acids.
  • R 1 , R 2 and R 3 can be substituted with hydroxyl and/or epoxy groups and/or can contain triple bonds.
  • the polyunsaturated fatty acids produced in the process according to the invention advantageously contain at least two, preferably three, four, five or six double bonds. It is particularly advantageous for the fatty acids to contain four, five or six double bonds.
  • Fatty acids produced in the process advantageously have 18, 20 or 22 carbon atoms in the fatty acid chain; the fatty acids preferably contain 20 or 22 carbon atoms in the fatty acid chain. Saturated fatty acids are advantageously reacted little or not at all with the nucleic acids used in the process.
  • the saturated fatty acids have less than 5% of the activity, advantageously less than 3%, particularly advantageously less than 2%, very particularly preferably less than 1; 0.5; 0.25 or 0.125% can be implemented.
  • These produced fatty acids can be produced as a single product in the process or can be present in a fatty acid mixture.
  • the substituents R 2 or R 3 in the general formulas I and II independently mean saturated or unsaturated C 18 -C 22 alkylcarbonyl, particularly advantageously they independently mean unsaturated C 18 , C 20 or C 22 alkylcarbonyl. with at least two double bonds.
  • ⁇ 12-desaturase sequences can be used alone or in combination with the ⁇ 3-desaturase sequences with the nucleic acid sequences according to the invention and not according to the invention used in the method, which are for ⁇ 9-elongases, ⁇ 6-desaturases, ⁇ 8-desaturases, ⁇ 6-elongases, ⁇ 5-desaturases, ⁇ 5-desaturases, Elongases and/or ⁇ 4-desaturases can be used.
  • Table 1 shows the nucleic acid sequences, the organism of origin and the sequence ID number. No. organism activity Sequence number 1. Euglena gracilis ⁇ 8-desaturase SEQ ID NO: 1 2.
  • Isochrysis galbana ⁇ 9-elongase SEQ ID NO: 3 Phaeodactylum tricornutum ⁇ 5-desaturase SEQ ID NO: 5 4. Ceratodon purpureus ⁇ 5-desaturase SEQ ID NO: 7 5. Physcomitrella patens ⁇ 5-desaturase SEQ ID NO: 9 6. Thraustrochytrium sp. ⁇ 5-desaturase SEQ ID NO: 11 7. Mortierella alpina ⁇ 5-desaturase SEQ ID NO: 13 8th. Caenorhabditis elegans ⁇ 5-desaturase SEQ ID NO: 15 9.
  • Ostreococcus tauri ⁇ 5-elongase SEQ ID NO: 75 37. Ostreococcus tauri ⁇ 5-elongase SEQ ID NO: 77 38. Ostreococcus tauri ⁇ 5-elongase SEQ ID NO: 79 39. Ostreococcus tauri ⁇ 6-elongase SEQ ID NO: 81 40. Thraustrochytrium sp. ⁇ 5-elongase SEQ ID NO: 83 41. Thalassiosira pseudonana ⁇ 5-elongase SEQ ID NO: 85 42.
  • Ostreococcus tauri ⁇ 5-elongase SEQ ID NO: 113 56.
  • Xenopus laevis BC044967
  • Ciona intestinalis ⁇ 5-elongase SEQ ID NO: 119
  • Euglena gracilis ⁇ 5-elongase SEQ ID NO: 131
  • Euglena gracilis ⁇ 5-elongase SEQ ID NO:133
  • Arabidopsis thaliana ⁇ 5-elongase SEQ ID NO: 135 61.
  • Arabidopsis thaliana ⁇ 5-elongase SEQ ID NO: 137 62. Phaeodactylum tricornutum ⁇ 6-elongase SEQ ID NO: 183
  • the polyunsaturated fatty acids produced in the process are advantageous in membrane lipids and/or Triacylglycerides bound, but can also occur in the organisms as free fatty acids or bound in the form of other fatty acid esters. They can be present as “pure products” or advantageously in the form of mixtures of different fatty acids or mixtures of different glycerides.
  • the different fatty acids bound in the triacylglycerides can be derived from short-chain fatty acids with 4 to 6 carbon atoms, medium-chain fatty acids with 8 to 12 carbon atoms or long-chain fatty acids with 14 to 24 carbon atoms; long-chain fatty acids are particularly preferred the long-chain fatty acids LCPUFAs of C 18 , C 20 and/or C 22 fatty acids.
  • Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are produced in the process according to the invention.
  • the fatty acid esters with polyunsaturated C 18 , C 20 and/or C 22 fatty acid molecules can be obtained from the organisms used for the production of the fatty acid esters in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, phosphoglycerides, lipids , glycolipids such as glycosphingolipids, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzyme A esters, which contain the polyunsaturated fatty acids with at least two, three, four, five or six, preferably contain five or six double bonds, are isolated, advantageously they are isolated in the form
  • the polyunsaturated fatty acids are also contained in plants as free fatty acids or bound in other compounds in the organisms.
  • the various aforementioned compounds are present in the organisms in an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of monoglycerides, 1 up to 5% by weight of free fatty acids, 2 to 8% by weight of phospholipids, with the sum of the various compounds adding up to 100% by weight.
  • the LCPUFAs produced have a content of at least 3% by weight, advantageously of at least 5% by weight, preferably of at least 8% by weight, particularly preferably of at least 10% by weight, very particularly preferably of at least 15% by weight based on the total fatty acids in the transgenic organisms are advantageously produced in a transgenic plant.
  • C 18 and/or C 20 fatty acids, which are present in the host organisms, are advantageously incorporated into the corresponding products to an extent of at least 10%, advantageously at least 20%, particularly advantageously at least 30%, and most advantageously at least 40% such as DPA or DHA, to name just two examples.
  • the fatty acids are advantageously produced in bound form.
  • these unsaturated fatty acids can be brought to the sn1, sn2 and/or sn3 position of the advantageously produced triglycerides.
  • the starting compounds linoleic acid (C 18:2 ) or linolenic acid (C 18:3 ) go through several reaction steps, the end products of the process such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), ⁇ 6-docosapentaenoic acid or DHA not as absolute pure products; there are always small traces of the precursors in the final product.
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA ⁇ 6-docosapentaenoic acid or DHA not as absolute pure products
  • the end products such as ARA, EPA or DHA are present as mixtures.
  • the precursors should advantageously not be more than 20% by weight, preferably not more than 15% by weight, particularly preferably not more than 10% by weight, very particularly preferably not more than 5% by weight, based on the amount of each Final product amount.
  • ARA, EPA or only DHA are bound as end products in a transgenic plant in the process according to the invention or produced as free acids. If the compounds ARA, EPA and DHA are produced simultaneously, they are advantageously in a ratio of at least 1:1:2 (EPA:ARA:DHA), advantageously at least 1:1:3, preferably 1:1:4, especially preferably made from 1:1:5.
  • Fatty acid esters or fatty acid mixtures that were produced by the process according to the invention advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid; 7 to 85% oleic acid; 0.5 to 8% vaccenic acid, 0.1 to 1% arachidic acid, 7 to 25% saturated fatty acids, 8 to 85% monounsaturated fatty acids and 60 to 85% polyunsaturated fatty acids, each based on 100% and on the total fatty acid content of the organisms.
  • Advantageous polyunsaturated fatty acids in the fatty acid esters or fatty acid mixtures are preferably at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; Contain 0.9 or 1% based on the total fatty acid content of arachidonic acid.
  • the fatty acid esters or fatty acid mixtures that were produced by the process according to the invention contain: advantageous fatty acids selected from the group of fatty acids erucic acid (13-docosaenoic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9-methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentene-dodecanoic acid), Furan fatty acid (9,12-epoxy-octadeca-9,11-dienonic acid), vernonic acid (9,10-epoxyoctadec-12-enonic acid), taric acid (6-octadecynonic acid), 6-nonadecynonic acid, santalbic acid (t11-octadecene-9 -ynoic acid), 6,9-octadecenynonic acid, pyrulic acid (t10-
  • the aforementioned fatty acids are usually only present in traces in the fatty acid esters or fatty acid mixtures produced by the process according to the invention, i.e. they occur in less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, particularly preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1%. In a further preferred form of the invention, these aforementioned fatty acids make up less than 0.9% of the total fatty acids; 0.8%; 0.7%; 0.6%; or 0.5%, particularly preferably less than 0.4%; 0.3%; 0.2%; 0.1% before.
  • the nucleic acid sequences used in the method according to the invention can increase the yield of polyunsaturated fatty acids by at least 50%, advantageously by at least 80%, particularly advantageously by at least 100%, very particularly advantageously by at least 150% compared to the non-transgenic starting organism, for example a yeast Algae, a fungus or a plant such as Arabidopsis or flax can be achieved when compared in the GC analysis see examples.
  • Chemically pure polyunsaturated fatty acids or fatty acid compositions can also be prepared using the methods described above.
  • the fatty acids or the fatty acid compositions are extracted from the non-human organism such as the microorganisms or the plants or the culture medium in or on which the organisms were grown, or from the organism and the culture medium in a known manner, for example via extraction, distillation, crystallization , chromatography or combinations of these methods.
  • These chemically pure fatty acids or fatty acid compositions are suitable for applications in the food industry, the cosmetics industry and especially the pharmaceutical industry.
  • non-human organisms such as microorganisms, non-human animals or plants come into question as organisms for production in the process according to the invention.
  • plants that are able to synthesize fatty acids can be considered as plants.
  • Advantageous plants are selected from the group of plant families Adelotheciaceae, Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulaceae, Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae , Juglandaceae, Lauraceae, Leguminosae, Linaceae, Euglenaceae, Prasinophyceae or vegetable plants or ornamental plants such as marigolds.
  • the following plants are selected from the group: Adelotheciaceae such as the genera Physcomitrella, for example the genus and species Physcomitrella patens, Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium, for example the genus and species Pistacia vera [pistachio], Mangifer indica [mango] or Anacardium occidentale [Cashew], Asteraceae such as the genera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana e.g.
  • Brassicaceae such as the genera Brassica, Camelina, Melanosinapis, Sinapis, Arabadopsis e.g. the genera and species Brassica napus, Brassica rapa ssp. [rapeseed], Sinapis arvensis Brassica juncea, Brassica juncea var. juncea, Brassica juncea var , Bromeliaceae such as the genera Anana, Bromelia (pineapple) e.g.
  • Anana comosus Ananas ananas or Bromelia comosa [pineapple]
  • Caricaceae such as the genus Carica such as the genus and species Carica papaya [Papaya]
  • Cannabaceae such as the genus Cannabis such as the genus and species Cannabis sative [hemp]
  • Convolvulaceae such as the genera Ipomea, Convolvulus e.g.
  • Cymbellaceae such as the genera Amphora, Cymbella, Okedenia, Phaeodactylum, Reimeria e.g. the genus and species Phaeodactylum tricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis, Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium, Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon, Skottsbergia e.g.
  • Ceratodon antarcticus Ceratodon columbiae, Ceratodon heterophyllus, Ceratodon purpurascens, Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon purpureus ssp.
  • genus and species Olea europaea [Olive] such as the genus Kalmia e.g. the genera and species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros or Kalmia lucida [mountain laurel], Euglenaceae such as the genera Ascoglena, Astasia, Colacium, Cyclidiopsis, Euglena, Euglenopsis, Hyalaphacus , Khawkinea, Lepocinclis, Phacus, Strombomonas, Trachelomonas e.g.
  • Euphorbiaceae such as the genera Manihot, Janipha, Jatropha, Ricinus e.g. the genera and species Manihot utilissima, Janipha manihot, Jatropha manihot., Manihot aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta [Manihot] or Ricinus communis [Castor bean], Fabaceae such as the genera Pisum, Albizia, Cathormion, Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseolus, Soja e.g.
  • Juglandaceae such as the genera Juglans, Wallia e.g. the genera and species Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans cinerea , Wallia cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra [walnut], Lauraceae Like the genera Persea, Laurus e.g.
  • Laurus nobilis [laurel ], Persea ameri-cana, Persea gratissima or Persea persea [Avocado], Leguminosae such as the genus Arachis e.g. the genus and species Arachis hypogaea [peanut], Linaceae such as the genera Linum, Adenolinum e.g.
  • Linum usitatissimum Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linum pratense or Linum trigynum [flax], Lythrarieae such as the genus Punica e.g. the genus and species Punica granatum [pomegranate], Malvaceae such as the genus Gossypium e.g.
  • Hordeum vulgare the genera and species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeum hexastichon., Hordeum hexastichum, Hordeum irregulare, Hordeum sativum, Hordeum secalinum [barley], Secale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.
  • Proteaceae such as the genus Macadamia e.g. the genus and species Macadamia intergrifolia [Macadamia ], Prasinophyceae such as the genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus e.g.
  • Verbascum blattaria Verbascum chaixii, Verbascum densiflorum, Verbascum lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum nigrum , Verbascum Olympicum, Verbascum Phlomoides, Verbascum Phoenicum, Verbascum Pulverulentum or Verbascum Thapsus [Königskerze], Solanaceae such as the genera Capsicum, Nicotiana, Solanum, Lycopersicon, for example, the genera and types capsicum annu, capsic.
  • Beneficial microorganisms are, for example, fungi selected from the group of families Chaetomiaceae, Choanephoraceae, Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae or Tuberculariaceae.
  • the following microorganisms are selected from the group: Choanephoraceae such as the genera Blakeslea, Choanephora e.g. the genera and species Blakeslea trispora, Choanephora cucurbitarum, Choanephora infundibulifera var. cucurbitarum, Mortierellaceae such as the genus Mortierella e.g. the genera and species Mortierella isabellina, Mortierella polycephala , Mortierella ramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae such as the genera Phytium, Phytophthora e.g.
  • Saccharomyces ellipsoideus Saccharomyces chevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomyces ellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus, Saccharomyces frag ilis, Saccharomyces heterogenicus, Saccharomyces hienipiensis, Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces kluyveri , Saccharomyces krusei, Saccharomyces lactis, Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomy
  • bacteria selected from the group of families Bacillaceae, Enterobacteriacae or Rhizobiaceae.
  • Bacillaceae such as the genus Bacillus, for example the genera and species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus sphaericus subsp. fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus globisporus subsp.
  • Bacillaceae such as the genus Bacillus, for example the genera and species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus spha
  • Bacillus halophilus Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus psychrosaccharomicus, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp. subtilis or Bacillus thuringiensis; Enterobacteriacae such as the genera Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella or Serratia e.g.
  • Citrobacter amalonaticus Citrobacter diversus, Citrobacter freundii, Citrobacter genomospecies, Citrobacter gillenii, Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp .
  • Edwardsiella hoshinae Edwardsiella ictaluri, Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwinia ananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia cacticida, Erwinia cancerogena, Erwinia carnegieana, Erwinia carotovora subsp.
  • marcescens Serratia marinorubra, Serratia odorifera, Serratia plymouthensis, Serratia plymuthica, Serratia proteamaculans, Serratia proteamaculans subsp. quinovora, Serratia quinivorans or Serratia rubidaea; Rhizobiaceae such as the genera Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium e.g.
  • microorganisms for the method according to the invention are, for example, protists or diatoms selected from the group of the families Dinophyceae, Turaniellidae or Oxytrichidae such as the genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina, Stylonychia notophora, Stylonychia sp. , Colpidium campylum or Colpidium sp.
  • the method according to the invention advantageously uses transgenic non-human organisms such as fungi such as Mortierella or Traustochytrium, yeasts such as Saccharomyces or Schizosaccharomyces, mosses such as Physcomitrella or Ceratodon, non-human animals such as Caenorhabditis, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium or Phaeodactylum or plants such as dicots or monocots are used.
  • fungi such as Mortierella or Traustochytrium
  • yeasts such as Saccharomyces or Schizosaccharomyces
  • mosses such as Physcomitrella or Ceratodon
  • non-human animals such as Caenorhabditis
  • algae such as Nephroselmis, P
  • organisms which belong to the oil-producing organisms i.e. which are used for the production of oils
  • oils such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum or plants, especially plants preferably oil crop plants that contain large amounts of lipid compounds, such as peanut, rapeseed, canola, sunflower, safflower (Carthamus tinctoria), poppy, mustard, hemp, castor bean, olive, sesame, calendula, punica , evening primrose, mullein, thistle, wild rose, hazelnut, almond, macadamia, avocado, laurel, pumpkin, flax, soy, pistachio, borage,
  • fungi such as Mortierella or
  • Preferred plants according to the invention are oil fruit plants, such as peanut, rapeseed, canola, sunflower, safflower, poppy, mustard, hemp, castor, olive, calendula, punica, evening primrose, pumpkin, linseed, soy, borage, trees (oil palm, coconut).
  • Plants rich in C 18:2 and/or C 18:3 fatty acids such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower are particularly preferred.
  • Plants such as safflower, sunflower, poppy, evening primrose, walnut, flax or hemp are particularly preferred.
  • nucleic acids introduced in process steps (a) to (d) and the possibly introduced nucleic acid sequences which code for the ⁇ 3-desaturases additional nucleic acids which are for enzymes of the fatty acid or encode lipid metabolism.
  • genes of fatty acid or lipid metabolism can be advantageously combined with the inventive ⁇ 5-elongase(s), ⁇ 6-elongase(s) and/or ⁇ 3-desaturase(s) [in the sense of this application the plural should be the Singular and vice versa include] are used in the process for producing polyunsaturated fatty acids.
  • Genes are particularly preferably selected from the group of ⁇ 4-desaturases, ⁇ 5-desaturases, ⁇ 6-desaturases, ⁇ 8-desaturases, ⁇ 9-desaturases, ⁇ 12-desaturases, ⁇ 6-elongases or ⁇ 9-elongases in combination with the aforementioned genes for the ⁇ 5-desaturases.
  • Elongase, ⁇ 6-elongase and/or ⁇ 3-desaturase are used, whereby individual genes or several genes can be used in combination.
  • the ⁇ 5-elongases Compared to human elongases or elongases from non-human animals such as those from Oncorhynchus, Xenopus or Ciona, the ⁇ 5-elongases have the advantageous property that they do not elongate C22 fatty acids to the corresponding C24 fatty acids. Furthermore, they advantageously do not convert fatty acids with a double bond in the ⁇ 6 position, as are converted by human elongases or elongases from non-human animals. Particularly advantageous ⁇ 5-elongases preferentially only convert unsaturated C20 fatty acids. These advantageous ⁇ 5-elongases have several putative transmembrane helixes (5-7).
  • C20 fatty acids with a double bond in the ⁇ 5 position are converted, with ⁇ 3-C20 fatty acids being preferred (EPA).
  • EPA ⁇ 3-C20 fatty acids
  • they have the property that, in addition to the ⁇ 5-elongase activity, they advantageously have no or only a relatively low ⁇ 6-elongase activity.
  • the human elongases or non-human animal elongases have approximately the same activity towards fatty acids with a ⁇ 6 or ⁇ 5 double bond. These advantageous elongases are referred to as so-called monofunctional elongases.
  • the human elongases or the non-human animal longases are referred to as multifunctional elongases, which, in addition to the aforementioned substrates, also convert monounsaturated C16 and C18 fatty acids, for example with ⁇ 9 or ⁇ 11 double bonds.
  • less than 60% by weight of the added GLA is converted to dihomo- ⁇ -linolenic acid (C20:3 ⁇ 8,11,14), advantageously less than 55% by weight, preferably less than 50% by weight. %, particularly advantageously less than 45% by weight, very particularly advantageously less than 40% by weight.
  • GLA is not converted.
  • FIGs 27 and 28 show the measured substrate specificities of the various elongases.
  • Figure 27 are the specificities of the multifunctional elongases from Xenopus laevis ( Figure 27A ), Ciona intestinalis ( Figure 27B ) and Oncorhynchus mykiss ( Figure 27C ) played. All of these elongases convert a wide range of substrates. In the process according to the invention, this can lead to by-products that have to be converted by further enzymatic activities. These enzymes are therefore less preferred in the process according to the invention.
  • the preferred monofunctional elongases and their substrate specificity are discussed in Figure 28 reproduced.
  • Figure 28A shows the specificity of the Ostreococcus tauri ⁇ 5-elongase. This only converts fatty acids with a double bond in the ⁇ 5 position. Only C 20 fatty acids are advantageously converted.
  • the ⁇ 5-elongase from Thalassiosira pseudonana ( Figure 28C ) on.
  • Both the ⁇ 6-elongase from Ostreococcus tauri ( Figure 28B ) as well as that of Thalassiosira pseudonana ( Figure 28D ) advantageously only convert fatty acids with a double bond in the ⁇ 6 position. Only C 18 fatty acids are advantageously converted.
  • the ⁇ 5-elongases from Arabidopsis thaliana and Euglena gracilis are also characterized by their specificity.
  • the ⁇ 6 elongases described are also characterized by high specificity, i.e. C 18 fatty acids are preferably elongated. They advantageously convert fatty acids with a double bond in the ⁇ 6 position. Particularly advantageous ⁇ 6 elongases advantageously convert C 18 fatty acids with three or four double bonds in the molecule, which must contain a double bond in the ⁇ 6 position. Furthermore, at best they have the property that, in addition to the ⁇ 6-elongase activity, they advantageously have no or only a relatively low ⁇ 5-elongase activity. In contrast, the human elongases or non-human animal elongases have approximately the same activity towards fatty acids with a ⁇ 6 or ⁇ 5 double bond.
  • the human elongases or the non-human animal longases are, as described above, referred to as multifunctional elongases, which, in addition to the aforementioned substrates, also convert mono-unsaturated C 16 and C 18 fatty acids, for example with ⁇ 9 or ⁇ 11 double bonds.
  • Particularly good ⁇ 6 elongases show no or very little activity (less than 0.1% by weight conversion) towards the following substrates: C 18:1 ⁇ 6 , C 18:1 ⁇ 9 , C 18: ⁇ 11 , C 20:2 ⁇ 11,14 , C 20:3 ⁇ 11,14,17 , C 20:3 ⁇ 8,11,14 , C 20:4 ⁇ 5,8,11,14 , C 20:5 ⁇ 5,8,11,14,17 or C 22:4 ⁇ 7,10,13,16 .
  • the Figures 29 and 30 and Table 18 show the measured substrate specificities of the various elongases.
  • the disclosed ⁇ 3-desaturase has the advantageous property that it can desaturate a wide range of ⁇ 6 fatty acids; C 20 and C 22 fatty acids such as C 20:2 , C 20:3 are preferred. , C 20:4 , C 22:4 or C 22:5 fatty acids desaturated. But the shorter C 18 fatty acids such as C 18:2 or C 18:3 fatty acids are also desaturated well. These properties of the ⁇ 3-desaturase make it possible to shift the fatty acid spectrum within an organism, ideally within a plant or a fungus, from the ⁇ 6 fatty acids to the ⁇ 3 fatty acids.
  • the described ⁇ 3-desaturase preferably desaturates C 20 fatty acids. Within the organism, at least 10%, 15%, 20%, 25% or 30% of these fatty acids from the existing fatty acid pool are converted into the corresponding ⁇ 3 fatty acids. Compared to the C 18 fatty acids, the ⁇ 3-desaturase has a lower activity by a factor of 10, which means that only approx. 1.5 to 3% of the fatty acids present in the fatty acid pool are converted into the corresponding ⁇ 3 fatty acids.
  • Preferred substrate of the invention ⁇ 3-desaturase are the ⁇ 6 fatty acids bound in phospholipids.
  • PC phosphatidylcholine
  • PIS phosphatidylinositol
  • PE phosphatidylethanolamine
  • the ⁇ 4-desaturases, ⁇ 5-desaturases and ⁇ 6-desaturases described have the advantage over the known ⁇ 4-desaturases, ⁇ 5-desaturases and ⁇ 6-desaturases in that they can convert fatty acids bound to phospholipids or CoA fatty acid esters, advantageously CoA fatty acid esters.
  • the ⁇ 12-desaturases used advantageously convert fatty acids bound to phospholipids or CoA fatty acid esters, advantageously bound to CoA fatty acid esters.
  • nucleic acids used in the method according to the invention which code for polypeptides with ⁇ 5-elongase, ⁇ 6-elongase and/or ⁇ 3-desaturase activity, advantageously in combination with nucleic acid sequences which code for polypeptides of fatty acid or lipid metabolism such as other polypeptides Encoding ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 8, ⁇ 12 desaturase or ⁇ 5, ⁇ 6 or ⁇ 9 elongase activity, a wide variety of polyunsaturated fatty acids can be produced in the process according to the invention.
  • mixtures of the various polyunsaturated fatty acids or individual polyunsaturated fatty acids such as EPA or ARA can be produced in free or bound form.
  • fatty acids are created that are derived from C 18:2 fatty acids, such as GLA, DGLA or ARA or that are derived from C 18 :3 fatty acids, such as SDA, ETA or EPA.
  • the only products that can be formed as products of the process are GLA, DGLA and ARA, which can be present as free fatty acids or bound.
  • ⁇ 5-elongase involved in the synthesis advantageous in combination with the ⁇ 4-, ⁇ 5-, ⁇ 6-, ⁇ 12-desaturase and/or ⁇ 6-elongase, or the ⁇ 4-, ⁇ 5-, ⁇ 8-, ⁇ 12- Desaturase and/or ⁇ 9-elongase can be specifically produced in the aforementioned organisms, advantageously only in individual products in the aforementioned plants.
  • the activity of ⁇ 6-desaturase and ⁇ 6-elongase produces, for example, GLA and DGLA or SDA and ETA, depending on the source plant and unsaturated fatty acid.
  • DGLA or ETA or mixtures thereof are preferably formed. If the ⁇ 5-desaturase, the ⁇ 5-elongase and the ⁇ 4-desaturase are also advantageously introduced into the organisms in the plant, ARA, EPA and/or DHA are also formed. This also applies to organisms into which the ⁇ 8-desaturase and ⁇ 9-elongase were previously introduced.
  • ARA, EPA or DHA or mixtures thereof are synthesized, depending on the fatty acid present in the organism or plant, which serves as the starting substance for the synthesis. Since these are biosynthesis chains, the respective end products are not present as pure substances in the organisms. Small amounts of the precursor compounds are always contained in the end product.
  • the protein encoded by the nucleic acid shows a high specificity for the two precursors C 18:4 ⁇ 6,9,12,15 - and C 20:5 ⁇ 5,8,11,14,17 - fatty acids for the synthesis of DHA (precursors and synthesis of DHA see Figure 1 ).
  • the protein encoded by SEQ ID NO: 53 therefore has specificity for ⁇ 6 and ⁇ 5 fatty acids with an additional ⁇ 3 double bond ( Figure 2 ).
  • the ⁇ 5-elongase has keto-acyl-CoA synthase activity that beneficially extends fatty acid residues of acyl-CoA esters by two carbon atoms.
  • the fatty acids can also be fed from outside. For cost reasons, production in the organism is preferred.
  • the preferred substrates of ⁇ 3-desaturase are linoleic acid (C 18:2 ⁇ 9.12 ), ⁇ -linolenic acid (C 18:3 ⁇ 6,9,12 ), eicosadienoic acid (C 20:2 ⁇ 11,14 ), dihomo- ⁇ -linolenic acid (C 20:3 ⁇ 8,11,14 ), arachidonic acid (C 20:4 ⁇ 5,8,11,14 ), docosatetraenoic acid (C 22:4 ⁇ 7,10,13,16 ) and docosapentaenoic acid ( C 22:5 ⁇ 4,7,10,13,15 ).
  • the genus and species Olea europaea or the family Fabaceae such as the genus Glycine e.g. the genus and species Glycine max, which have a high oleic acid content. Since these organisms only have a low content of linoleic acid ( Mikoklajczak et al., Journal of the American Oil Chemical Society, 38, 1961, 678 - 681 ), the use of the ⁇ 12-desaturases mentioned is advantageous for producing the starting product linoleic acid.
  • Nucleic acids used in the method according to the invention advantageously come from plants such as algae, for example algae of the Prasinophyceae family such as from the genera Heteromastix, Mammella, Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda, Pycnococcus, Pyramimonas, Scherffelia or Tetraselmis such as the genera and Species Heteromastix longifillis, Mamiella gilva, Mantoniella squamata, Micromonas pusilla, Nephroselmis olivacea, Nephroselmis pyriformis, Nephroselmis rotunda, Ostreococcus tauri, Ostreococcus sp.
  • algae of the Prasinophyceae family such as from the genera Heteromastix,
  • the nucleic acids used advantageously come from algae of the genera Euglena, Mantoniella or Ostreococcus.
  • algae such as Isochrysis or Crypthecodinium
  • algae/diatoms such as Thalassiosira or Phaeodactylum
  • mosses such as Physcomitrella or Ceratodon or higher plants such as the Primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides
  • microorganisms such as fungi such as Aspergillus, Thraustochytrium, Phytophthora , Entomophthora, Mucor or Mortierella, bacteria like Shewanella, yeasts or animals like nematodes like Caenorhabditis, insects, frogs, sea cucumbers or fish.
  • the isolated nucleic acid sequences according to the invention advantageously come from an animal from the vertebrate order.
  • the nucleic acid sequences preferably come from the class Vertebrata; Euteleostomi, Actinopterygii; Neopterygii; teleost egg; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus or Vertebrata, Amphibia, Anura, Pipidae,
  • the nucleic acids come particularly advantageously from fungi, animals or from plants such as algae or mosses, preferably from the order Salmoniformes such as the family Salmonidae such as the genus Salmo, for example from the genera and species Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario, from algae such as the genera Mantoniella or Ostreococcus or from the diatoms such as the genera Thalass
  • nucleic acid sequences or their derivatives or homologues which code for polypeptides which still have the enzymatic activity of the proteins encoded by nucleic acid sequences.
  • These sequences are cloned into expression constructs individually or in combination with the nucleic acid sequences encoding the ⁇ 12-desaturase, ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 5-elongase, ⁇ 6-elongase and / or ⁇ 3-desaturase and for introduction and use Expression used in organisms. Due to their construction, these expression constructs enable an advantageous, optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
  • the method further comprises the step of obtaining a cell or an entire organism which contains the nucleic acid sequences used in the method, the cell and/or the organism being provided with a nucleic acid sequence according to the invention which codes for the A5 elongase, a gene construct or a vector as described below, alone or in combination with further nucleic acid sequences that code for proteins of fatty acid or lipid metabolism.
  • this method further comprises the step of recovering the oils, lipids or free fatty acids from the organism or from the culture.
  • the culture can be, for example, a fermentation culture, for example in the case of the cultivation of microorganisms such as Mortierella, Thalassiosira, Mantoniella, Ostreococcus, Saccharomyces or Thraustochytrium, or a greenhouse or field culture of a plant.
  • the cell or organism so produced is advantageously a cell of an oil-producing organism such as an oil crop plant such as peanut, rapeseed, canola, flax, hemp, soy, safflower, sunflower or borage.
  • Cultivation is, for example, the cultivation of plant cells, tissues or organs on or in a nutrient medium or the entire plant on or in a substrate, for example in hydroponics, potting soil or on arable land.
  • transgenic organism or transgenic plant in the context of the invention means that the nucleic acids used in the method are not in their natural location in the genome of an organism; the nucleic acids can be expressed homologously or heterologously.
  • transgenic also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and/or that the regulatory sequences of the natural sequences have been changed.
  • Transgenic is preferably understood to mean the expression of the nucleic acids according to the invention at a non-natural location in the genome, that is to say there is a homologous or preferably heterologous expression of the nucleic acids.
  • Preferred transgenic organisms are fungi such as Mortierella or Phytophtora, mosses such as Physcomitrella, algae such as Mantoniella, Euglena, Crypthecodinium or Ostreococcus, diatoms such as Thalassiosira or Phaeodyctylum or plants such as the oil fruit plants.
  • fungi such as Mortierella or Phytophtora
  • mosses such as Physcomitrella
  • algae such as Mantoniella, Euglena, Crypthecodinium or Ostreococcus
  • diatoms such as Thalassiosira or Phaeodyctylum or plants such as the oil fruit plants.
  • all organisms that are able to synthesize fatty acids, especially unsaturated fatty acids, or are suitable for the expression of recombinant genes are advantageously suitable as organisms or host organisms for the nucleic acids used in the method according to the invention, the expression cassette or the vector.
  • Examples include plants such as Arabidopsis, Asteraceae such as Calendula or cultivated plants such as soy, peanut, castor bean, sunflower, corn, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophtora or Pythium, bacteria such as the genus Escherichia or Shewanella, yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae such as Mantoniella, Euglena, Thalassiosira or Ostreococcus or protozoa such as dinoflagellates such as Crypthecodinium.
  • fungi for example the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophtora
  • transgenic animals are also advantageously suitable as host organisms: non-human animals, for example C. elegans, Ciona intestinalis or Xenopus laevis.
  • Transgenic plants that contain the polyunsaturated fatty acids synthesized in the process according to the invention can advantageously be marketed directly without the synthesized oils, lipids or fatty acids having to be isolated.
  • Plants in the method according to the invention include whole plants as well as all plant parts, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, calli, cotelydons, petioles, harvest material, plant tissue, reproductive tissue, Cell cultures that are derived from the transgenic plant and/or can be used to produce the transgenic plant.
  • the seed includes all parts of the seed such as the seed coats, epidermis and sperm cells, endosperm or embryo tissue.
  • the compounds produced in the process according to the invention can also be isolated from the organisms, advantageously plants, in the form of their oils, fat, lipids and/or free fatty acids.
  • Polyunsaturated fatty acids produced by this process can be harvested by harvesting the organisms either from the crop in which they grow or from the field. This can be done by pressing or extracting the plant parts, preferably the plant seeds.
  • the oils, fats, lipids and/or free fatty acids can be obtained by so-called cold beating or cold pressing without the addition of heat by pressing.
  • they are first crushed, steamed or roasted.
  • the seeds pretreated in this way can then be pressed or extracted with a solvent such as warm hexane.
  • the solvent is then removed again.
  • these are extracted, for example, directly after harvest without any further work steps or extracted after digestion using various methods known to those skilled in the art. In this way, more than 96% of the compounds produced in the process can be isolated.
  • the products obtained in this way are then further processed, i.e. refined.
  • the plant mucilage and cloudy substances are removed.
  • the so-called degumming can be carried out enzymatically or, for example, chemically/physically by adding acid such as phosphoric acid.
  • the free fatty acids are then removed by treatment with a base, for example caustic soda.
  • the product obtained is thoroughly washed with water and dried to remove the lye remaining in the product.
  • the products are subjected to bleaching with, for example, bleaching earth or activated carbon.
  • the product is deodorized, for example with steam.
  • the PUFAs or LCPUFAs produced by this process are preferably C 18 , C 20 or C 22 fatty acid molecules, advantageously C 20 or C 22 fatty acid molecules with at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds.
  • These C 18 , C 20 or C 22 fatty acid molecules can be isolated from the organism in the form of an oil, lipid or a free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.
  • An embodiment of the invention is therefore oils, lipids or fatty acids or fractions thereof which have been produced by the method described above, particularly preferably oil, lipid or a fatty acid composition which comprises PUFAs and originates from transgenic plants.
  • these oils, lipids or fatty acids advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid; 7 to 85% oleic acid; 0.5 to 8% vaccenic acid, 0.1 to 1% arachidic acid, 7 to 25% saturated fatty acids, 8 to 85% monounsaturated fatty acids and 60 to 85% polyunsaturated fatty acids, each based on 100% and on the total fatty acid content of the organisms.
  • Advantageous polyunsaturated fatty acids in the fatty acid esters or fatty acid mixtures are preferably at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; Contain 0.9 or 1% based on the total fatty acid content of arachidonic acid.
  • the fatty acid esters or fatty acid mixtures that were produced by the process according to the invention advantageously contain fatty acids selected from the group of fatty acids: erucic acid (13-docosaenoic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8.9 -Methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentene-dodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,11-dienoic acid), vernonic acid (9,10-epoxyoctadec-12-enoic acid), taric acid ( 6-octadecynonic acid), 6-nonadecynonic acid, santalbic acid (t11-octadecene-9-ynoic acid), 6,9-octadecenynonic acid, pyrulinic acid (13
  • the aforementioned fatty acids are usually only present in traces in the fatty acid esters or fatty acid mixtures produced by the process according to the invention, i.e. they occur in less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, particularly preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1%. In a further preferred form of the invention, these aforementioned fatty acids come based on the Total fatty acids less than 0.9%; 0.8%; 0.7%; 0.6%; or 0.5%, particularly preferably less than 0.4%; 0.3%; 0.2%; 0.1% before.
  • oils, lipids or fatty acids according to the invention advantageously contain at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, 7%, 8%, 9% or 10%, particularly advantageously at least 11%, 12%, 13%, 14% or 15% ARA or at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, or 7%, particularly advantageously at least 8 %, 9% or 10% EPA and/or DHA based on the total fatty acid content of the production organism advantageously in a plant, particularly advantageously in an oil crop plant such as soy, rapeseed, coconut, oil palm, safflower, flax, hemp, castor bean, calendula, peanut, cocoa bean, sunflower or the other monocotyledonous or dicotyledonous oil crop plants mentioned above.
  • an oil crop plant such as soy, rapeseed, coconut, oil palm, safflower, flax, hemp, castor
  • the oil, lipid, fatty acids and/or fatty acid composition are used, for example, in feed, food, cosmetics or pharmaceuticals.
  • the oils, lipids, fatty acids or fatty acid mixtures can be used in the manner known to those skilled in the art for mixing with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, such as fish oils.
  • These oils, lipids, fatty acids or fatty acid mixtures, which consist of plant and animal components, can also be used to produce feed, food, cosmetics or pharmaceuticals.
  • oil is understood to mean a fatty acid mixture that contains unsaturated or saturated, preferably esterified fatty acid(s). It is preferred that the oil, lipid or fat contains a high proportion of polyunsaturated free or advantageously esterified fatty acid(s), in particular linoleic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, Docosapentaenoic acid or docosahexaenoic acid.
  • linoleic acid ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, Docosapent
  • the proportion of unsaturated esterified fatty acids is approximately 30%, more preferably a proportion of 50%, even more preferred a proportion of 60%, 70%, 80% or more.
  • the proportion of fatty acid can be determined by gas chromatography after the fatty acids have been converted into the methyl esters by transesterification.
  • the oil, lipid or fat may contain various other saturated or unsaturated fatty acids, for example calendulic acid, palmitic, palmitoleic, stearic, oleic acid, etc.
  • the proportion of different fatty acids in the oil or fat can vary depending on the source organism.
  • the polyunsaturated fatty acids produced in the process which should contain at least two double bonds, are, as described above, for example sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
  • the polyunsaturated fatty acids contained can be obtained, for example, via an alkali treatment, for example aqueous KOH or NaOH or acid hydrolysis, advantageously in the presence of an alcohol such as methanol or ethanol or via enzymatic cleavage release and isolate via, for example, phase separation and subsequent acidification via, for example, H 2 SO 4 .
  • the fatty acids can also be released directly without the processing described above.
  • the nucleic acids used in the method can advantageously be located in a plant cell or plant either on a separate plasmid or advantageously integrated into the genome of the host cell.
  • the integration may be random, or may occur by recombination such that the native gene is replaced by the introduced copy, thereby modulating the cell's production of the desired compound, or by using a gene in trans, so that the Gene is functionally linked to a functional expression unit which contains at least one sequence ensuring the expression of a gene and at least one sequence ensuring the polyadenylation of a functionally transcribed gene.
  • the nucleic acids are advantageously brought into the organisms via multiexpression cassettes or constructs for multiparallel expression of genes in the organisms for multiparallel seed-specific expression of genes.
  • Mosses and algae are the only known plant systems that produce significant amounts of polyunsaturated fatty acids such as arachidonic acid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA).
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • Mosses contain PUFAs in membrane lipids while algae, algae-related organisms and some fungi also accumulate significant amounts of PUFAs in the triacylglycerol fraction.
  • nucleic acid molecules that are isolated from strains that also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the method according to the invention and thus for modifying the lipid and PUFA production system in a host, in particular plants, such as oil crop plants, for example rapeseed, Canola, flax, hemp, soy, sunflower, borage. They can therefore be used advantageously in the process according to the invention.
  • the polyunsaturated C 18 fatty acids must first be desaturated by the enzymatic activity of a desaturase and then extended by at least two carbon atoms using an elongase. After one round of elongation, this enzyme activity leads to C 20 fatty acids, and after two rounds of elongation to C 22 fatty acids.
  • the activity of the desaturases and elongases used according to the invention preferably leads to C 18 , C 20 and/or C 22 fatty acids with at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, particularly preferably to C 20 and/or C 22 fatty acids with at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, very particularly preferably with five or six double bonds in the molecule.
  • Particularly preferred products of the process according to the invention are dihomo- ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapetaenoic acid and/or docosahesaenoic acid.
  • the C 20 fatty acids with at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
  • the preferred site of biosynthesis of fatty acids, oils, lipids or fats in the plants used advantageously is, for example, generally the seed or cell layers of the seed, so that seed-specific expression of the nucleic acids used in the process makes sense.
  • biosynthesis of fatty acids, oils or lipids does not have to be limited to the seed tissue, but can also take place in a tissue-specific manner in all other parts of the plant - for example in epidermal cells or in the tubers.
  • microorganisms such as yeasts such as Saccharomyces or Schizosaccharomyces
  • fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium algae such as Isochrysis, Mantoniella, Euglena, Ostreococcus, Phaeodactylum or Crypthecodinium are used as organisms in the process according to the invention, these organisms are advantageously grown by fermentation.
  • the polyunsaturated fatty acids produced in the process can be increased by at least 5%, preferably at least 10%, particularly preferably at least 20%, very particularly preferably at least 50% compared to the wild type of organisms that do not contain the nucleic acids recombinantly can be increased.
  • the polyunsaturated fatty acids produced in the organisms used in the process can in principle be increased in two ways.
  • the pool of free polyunsaturated fatty acids and/or the proportion of esterified polyunsaturated fatty acids produced via the process can advantageously be increased.
  • the process according to the invention advantageously increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms.
  • microorganisms are used as organisms in the process according to the invention, they are grown or grown in a manner known to those skilled in the art, depending on the host organism.
  • Microorganisms are usually grown in a liquid medium that contains a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and, if necessary, vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C and 60 ° C under oxygen gassing.
  • the pH of the nutrient liquid can be kept at a fixed value, which means it can be regulated or not during cultivation.
  • Cultivation can be done batchwise, semi-batchwise or continuously. Nutrients can be introduced at the beginning of fermentation or replenished semi-continuously or continuously.
  • the polyunsaturated fatty acids produced can be isolated from the organisms using methods known to those skilled in the art as described above. For example, via extraction, distillation, crystallization, possibly salt precipitation and/or chromatography. The organisms can be advantageously broken down beforehand.
  • the process according to the invention is advantageously carried out at a temperature between 0°C to 95°C, preferably between 10°C to 85°C, particularly preferably between 15°C to 75°C particularly preferably carried out between 15 ° C and 45 ° C.
  • the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, particularly preferably between pH 7 and 8.
  • the process according to the invention can be operated batchwise, semi-batchwise or continuously.
  • a summary of well-known cultivation methods is in the textbook by Chmiel (Bioprocess Engineering 1. Introduction in bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991 )) or in the textbook by Storhas (bioreactors and peripheral devices (Vieweg Verlag, Braunschweig/Wiesbaden, 1994 )) to find.
  • the culture medium to be used must suit the requirements of the respective strains. Descriptions of culture media of various microorganisms are in the manual " “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington DC, USA, 1981 ) contain.
  • these media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
  • Preferred carbon sources are sugars such as mono-, di- or polysaccharides.
  • sugars such as mono-, di- or polysaccharides.
  • very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugar can also be added to the media via complex compounds such as molasses or other byproducts of sugar refining. It may also be advantageous to add mixtures of different carbon sources.
  • oils and fats such as soybean oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as B. glycerin, methanol and / or ethanol and / or organic acids such as acetic acid and / or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
  • Exemplary nitrogen sources include ammonia in liquid or gas form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • Inorganic salt compounds that may be contained in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds such as mercaptans and thiols can be used as a sulfur source for the production of sulfur-containing fine chemicals, in particular methionine.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as a phosphorus source.
  • Chelating agents can be added to the medium to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
  • the fermentation media used according to the invention to cultivate microorganisms usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
  • Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like. Suitable precursors can also be added to the culture medium. The exact composition of the media compounds depends heavily on the respective experiment and is decided individually for each specific case. Information about media optimization is available from the textbook " Applied Microbiol. Physiology, A Practical Approach” (eds. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3 ). Growth media can also be obtained from commercial suppliers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
  • All media components are sterilized either by heat (20 min at 1.5 bar and 121 °C) or by sterile filtration.
  • the components can be sterilized either together or separately if necessary. All media components can be present at the beginning of cultivation or can be added continuously or in batches.
  • the temperature of the culture is normally between 15°C and 45°C, preferably 25°C to 40°C, and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
  • the pH value for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
  • anti-foam agents such as: B. fatty acid polyglycol esters can be used.
  • suitable selective substances can be added to the medium, such as: B. antibiotics can be added.
  • oxygen or gas mixtures containing oxygen such as ambient air, are introduced into the culture.
  • the temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C.
  • the culture is continued until a maximum of the desired product has been formed. This goal is typically achieved within 10 hours to 160 hours.
  • the fermentation broths obtained in this way in particular containing polyunsaturated fatty acids, usually have a dry matter of 7.5 to 25% by weight.
  • the fermentation broth can then be further processed.
  • the biomass can wholly or partly by separation methods, such as. B. centrifugation, filtration, decantation or a combination of these methods can be removed from the fermentation broth or left completely in it.
  • separation methods such as. B. centrifugation, filtration, decantation or a combination of these methods can be removed from the fermentation broth or left completely in it.
  • the biomass is advantageously processed after separation.
  • the fermentation broth can also be used without cell separation using known methods, such as. B. can be thickened or concentrated with the help of a rotary evaporator, thin film evaporator, falling film evaporator, reverse osmosis, or nanofiltration. This concentrated fermentation broth can finally be processed to obtain the fatty acids it contains.
  • the fatty acids obtained in the process are also suitable as starting material for the chemical synthesis of other valuable products.
  • they can be used in combination with each other or alone for the production of pharmaceuticals, food, animal feed or cosmetics.
  • nucleic acids with sequences that code for polypeptides with ⁇ 5-elongase activity, the ⁇ 5-elongases encoded by the nucleic acid sequences converting C20 fatty acids with at least four double bonds in the fatty acid molecule; which are ultimately advantageously incorporated into diacylglycerides and/or triacylglycerides.
  • nucleic acids with sequences which code for polypeptides with ⁇ 5-elongase activity and which contain an amino acid sequence selected from the group SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141 or SEQ ID NO: 142.
  • SEQ ID NO: 115 NXXXHXXMYXYYX
  • SEQ ID NO: 116 HHXXXXWAWW
  • SEQ ID NO: 139 LHXXHH
  • SEQ ID NO: 140 TXXQXXQF
  • SEQ ID NO: 141 DTXFMV
  • SEQ ID NO: 142 TQAQXXQF reproduced sequences represent conserved regions of the various elongases.
  • Table 2 shows the meaning of the amino acids marked with X contained in the nucleic acid sequences mentioned (column 3). The preferred amino acids in the different positions can also be found in the table (column 3).
  • Column 1 shows the SEQ ID NO, column 2 the position in the sequence.
  • Table 2 Meaning of the amino acid marked X in the consensus sequences.
  • Particularly advantageous ⁇ 5-elongases contain at least one of the sequences SEQ ID NO: 116, SEQ ID NO: 141 and/or SEQ ID NO: 142.
  • gene constructs which have the nucleic acid sequences SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63; SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO
  • acyl-CoA dehydrogenase(s)
  • Biosynthesis genes of fatty acid or lipid metabolism selected from the group of ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 8-desatuase, ⁇ 9-desaturase, ⁇ 12-desaturase, ⁇ 6-elongase, ⁇ 9-elongase or ⁇ 3-desaturase are also advantageously included .
  • nucleic acid sequences used in the method according to the invention come from a eukaryotic organism such as a plant, a microorganism or an animal.
  • the nucleic acid sequences preferably come from the order Salmoniformes,
  • the nucleic acid construct may contain more than one nucleic acid sequence of an enzymatic activity such as a ⁇ 12-desaturase, ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 5-elongase, ⁇ 6-elongase and/or ⁇ 3-desaturase.
  • an enzymatic activity such as a ⁇ 12-desaturase, ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 5-elongase, ⁇ 6-elongase and/or ⁇ 3-desaturase.
  • the nucleic acids used in the process are advantageously subjected to amplification and ligation in a known manner.
  • the procedure is based on the protocol of the Pfu DNA polymerase or a Pfu/Taq DNA polymerase mixture.
  • the primers are chosen based on the sequence to be amplified.
  • the primers should expediently be chosen so that the amplicon encompasses the entire codogenic sequence from the start to the stop codon.
  • the amplicon is expediently analyzed. For example, the analysis can be carried out after gel electrophoretic separation in terms of quality and quantity.
  • the amplicon can then be purified using a standard protocol (e.g. Qiagen).
  • Suitable cloning vectors are generally known to those skilled in the art. This includes, in particular, vectors that can be replicated in microbial systems, i.e. especially vectors that ensure efficient cloning in yeasts or fungi and enable the stable transformation of plants.
  • vectors that can be replicated in microbial systems, i.e. especially vectors that ensure efficient cloning in yeasts or fungi and enable the stable transformation of plants.
  • various binary and co-integrated vector systems suitable for T-DNA-mediated transformation should be mentioned. Such vector systems are usually characterized by the fact that they contain at least the vir genes required for the Agrobacterium-mediated transformation as well as the T-DNA limiting sequences (T-DNA border).
  • vector systems preferably also include further cis-regulatory regions such as promoters and terminators and/or selection markers with which correspondingly transformed organisms can be identified.
  • vir genes and T-DNA sequences are arranged on the same vector
  • binary systems are based on at least two vectors, one of which has vir genes but no T-DNA and a second T-DNA but no vir gene carries. This makes the latter vectors relatively small, easy to manipulate and replicate in both E. coli and Agrobacterium.
  • binary vectors include vectors from the pBIB-HYG, pPZP, pBecks, pGreen series.
  • the vectors can first be linearized with restriction endonuclease(s) and then enzymatically modified in a suitable manner. The vector is then purified and an aliquot is used for cloning. During cloning, the enzymatically cut and, if necessary, purified amplicon is ligated with similarly prepared vector fragments using ligase.
  • a specific nucleic acid construct or vector or plasmid construct can have one or more codogenic gene sections.
  • the codogenic gene sections in these constructs are preferably functionally linked to regulatory sequences.
  • the regulatory sequences particularly include plant ones Sequences such as the promoters and terminators described above.
  • the constructs can advantageously be stably propagated in microorganisms, in particular Escherichia coli and Agrobacterium tumefaciens, under selective conditions and enable transfer of heterologous DNA into plants or microorganisms.
  • nucleic acids used in the process can be introduced into organisms such as microorganisms or advantageously plants and can therefore be used in plant transformation, such as those published in and cited there: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993 ); FF White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Eds.: Kung and R. Wu, Academic Press, 1993, 15-38 ; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol.
  • nucleic acids used in the process can thus be advantageously used in plants for the genetic engineering modification of a broad spectrum of organisms, so that they become better and/or more efficient producers of PUFAs.
  • the number or activity of ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 9-elongase, ⁇ 6-desaturase, ⁇ 8-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase, or ⁇ 4-desaturase proteins or genes can be increased so that larger amounts of the gene products and thus ultimately larger amounts of the compounds of general formula I are produced.
  • a de novo synthesis in an organism that lacked the activity and ability to biosynthesize the compounds before the introduction of the corresponding gene(s) is also possible.
  • the number or activity of other genes involved in the import of nutrients necessary for the biosynthesis of one or more fatty acids, oils, polar and/or neutral lipids may be increased, so that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment is increased, further increasing the ability of cells to produce PUFAs as described below.
  • the isolated nucleic acid molecules used in the method according to the invention encode proteins or parts thereof, the proteins or the individual protein or parts thereof containing an amino acid sequence which is sufficiently homologous to an amino acid sequence which is represented in the sequence SEQ ID NO: 68, so that the proteins or parts thereof still have a ⁇ 5-elongase activity, the amino acid sequence not having the amino acid sequence shown in SEQ ID NO: 114.
  • the proteins or parts thereof that are encoded by the nucleic acid molecule(s) still have its essential enzymatic activity and the ability to participate in the metabolism of compounds necessary for the construction of cell membranes or lipid bodies in organisms or in the transport of molecules participate via these membranes.
  • the proteins encoded by the nucleic acid molecules are at least about 50%, preferably at least about 60% and more preferably at least about 70%, 80% or 90% and most preferably at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence shown in SEQ ID NO: 68, wherein the amino acid sequence is not has the amino acid sequence shown in SEQ ID NO: 114.
  • homology or homologous means identity or identical.
  • the proteins or parts thereof that are encoded by the nucleic acid molecule(s) still have its essential enzymatic activity and the ability to participate in the metabolism of compounds necessary for the construction of cell membranes or lipid bodies in organisms or in the transport of molecules participate via these membranes.
  • the proteins encoded by the nucleic acid molecules are at least about 50%, preferably at least about 60% and more preferably at least about 70%, 80% or 90% and most preferably at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
  • the homology was calculated over the entire amino acid or nucleic acid sequence range.
  • a number of programs based on different algorithms are available to those skilled in the art for comparing different sequences.
  • the algorithms from Needleman and Wunsch or Smith and Waterman deliver particularly reliable results.
  • the program PileUp was used for the sequence comparisons ( J. Mol. Evolution., 25, 351-360, 1987 , Higgins et al., CABIOS, 5 1989: 151-153 ) or the programs Gap and BestFit [ Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970 ) and Smith and Waterman (Adv. Appl. Math.
  • Substantial enzymatic activity of the ⁇ 5-elongase used in the method according to the invention is to be understood as meaning that it still has at least an enzymatic activity of at least 10%, preferably 20, compared to the proteins/enzymes encoded by the sequence with SEQ ID NO: 67 and its derivatives %, particularly preferably 30% and very particularly 40% and can therefore participate in the metabolism of compounds necessary for the construction of fatty acids, fatty acid esters such as diacylglycerides and / or triacylglycerides in an organism, advantageously a plant or plant cell, or in the transport of molecules across membranes, whereby C 18 -, C 20 - or C 22 -carbon chains in the fatty acid molecule with double bonds in at least two, advantageously three, four, five or six places are meant.
  • Nucleic acids that can advantageously be used in the method come from bacteria, fungi, diatoms, animals such as Caenorhabditis or Oncorhynchus or plants such as algae or mosses such as the genera Shewanella, Physcomitrella, Thraustochytrium, Fusarium, Phytophthora, Ceratodon, Mantoniella, Ostreococcus, Isochrysis, Aleurita, Muscarioides, Mortierella , Borago, Phaeodactylum, Crypthecodinium, especially from the genres and types oncorhynchus mykiss, Xenopus laevis, ciona intestinalis, thalassiosira pseudonona, mantoniella squamata, Ostrreococcus sp., Ostrtreococcus tauri, Euglena Gracilis, Physcomitrella, Physcomitrella Paten S
  • nucleotide sequences can be used in the method which are for a ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 9-elongase, ⁇ 6-desaturase, ⁇ 8-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase encode and which are attached to a nucleotide sequence, as in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 , SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
  • the nucleic acids used in the method are advantageously introduced into an expression cassette, which enables the expression of the nucleic acids in organisms such as microorganisms or plants.
  • nucleic acid sequences that code for the ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 9-elongase, ⁇ 6-desaturase, ⁇ 8-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase are coupled with one or more regulatory signals advantageously functionally linked to increase gene expression. These regulatory sequences are intended to enable the targeted expression of genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed and/or overexpressed after induction, or that it is expressed and/or overexpressed immediately.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences may still be present in front of the actual structural genes and may have been genetically modified so that the natural regulation was switched off and the expression of the genes was increased.
  • the gene construct can also advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3' end of the DNA sequences, such as further regulatory elements or terminators.
  • the expression cassette may contain only one copy of the genes.
  • This gene construct or the gene constructs can be expressed together in the host organism.
  • the gene construct or gene constructs can be inserted into one or more vectors and be present freely in the cell or inserted into the genome. It is advantageous for the insertion of additional genes into the host genome if the genes to be expressed are present together in a gene construct.
  • the regulatory sequences or factors can preferably positively influence and thereby increase the gene expression of the introduced genes.
  • the regulatory elements can thus advantageously be strengthened at the transcription level by using strong transcription signals such as “promoters” and/or “enhancers”.
  • promoters and/or “enhancers”.
  • SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO : 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 , SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO : 65, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO:
  • ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 9-elongase, ⁇ 6-desaturase, ⁇ 8-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase or ⁇ 4-desaturase proteins perform advantageously to desaturate or elongate fatty acids, the substrate advantageously having one, two, three, four, five or six double bonds and advantageously having 18, 20 or 22 carbon atoms in the fatty acid molecule.
  • Advantageous regulatory sequences for the new process are present, for example, in promoters such as the cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5 , T3, gal, trc, ara, SP6, ⁇ -PR or ⁇ -PL promoter and are advantageously used in Gram-negative bacteria.
  • promoters such as the cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5 , T3, gal, trc, ara, SP6, ⁇ -PR or ⁇ -PL promoter and are advantageously used in Gram-negative bacteria.
  • Further advantageous regulatory sequences are, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV/35S [ Franck et al., Cell21 (1980) 285-294 ], PRP1 [ Ward et al., Plant. Mol. Biol. 22 (1993 )], SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.
  • Inducible promoters such as those in, are also advantageous in this context EP-A-0 388 186 (Benzylsulfonamide-inducible), Plant J. 2, 1992:397-404 (Gatz et al., tetracycline-inducible), EP-A-0 335 528 (abcisic acid-inducible) or WO 93/21334 (Ethanol- or cyclohexenol-inducible) described promoters.
  • Other suitable plant promoters are the promoter of cytosolic FBPase or the ST-LSI promoter of the potato ( Stockhaus et al., EMBO J.
  • Particularly advantageous promoters are promoters that enable expression in tissues that are involved in fatty acid biosynthesis. Seed-specific promoters, such as the USP promoter according to the embodiment, but also other promoters such as the LeB4, DC3, Phaseolin or Napin promoter are particularly advantageous.
  • promoters are seed-specific promoters that can be used for monocotyledonous or dicotyledonous plants and in US 5,608,152 (Napin promoter from rapeseed), WO 98/45461 (Oleosin promoter from Arobidopsis), US 5,504,200 (Phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica), from Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4 promoter from a legume), these promoters being suitable for dicotyledons.
  • promoters are suitable for monocotyledons, for example Ipt-2 or Ipt-1 promoter from barley ( WO 95/15389 and WO 95/23230 ), hordein promoter from barley and others, in WO 99/16890 suitable promoters described.
  • the PUFA biosynthesis genes should be advantageously expressed in a seed-specific manner in oilseeds.
  • seed-specific promoters can be used, or promoters that are active in the embryo and/or in the endosperm. Seed-specific promoters can in principle be isolated from both dicotolydones and monocotolydones plants.
  • WO 95/23230 seed-specific promoters from rice, corn and wheat [ WO 99/16890 ], Amy32b, Amy 6-6 and Aleurain [ US 5,677,474 ], Bce4 (rapeseed) [ US 5,530,149 ], glycinin (soy) [ EP 571 741 ], phosphoenol pyruvate carboxylase (soy) [ JP 06/62870 ], ADR12-2 (soy) [ WO 98/08962 ], isocitrate lyase (rapeseed) [ US 5,689,040 ] or ⁇ -amylase (barley) [ EP 781 849 ].
  • Plant gene expression can also be facilitated via a chemically inducible promoter (see a review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108 ).
  • Chemically inducible promoters are particularly suitable when it is desired that gene expression occurs in a time-specific manner. Examples for such promoters are a salicylic acid-inducible promoter ( WO 95/19443 ), a tetracycline-inducible promoter ( Gatz et al. (1992) Plant J. 2, 397-404 ) and an ethanol-inducible promoter.
  • each of the nucleic acids used in the process which are responsible for the ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 9-elongase, ⁇ 6-desaturase, ⁇ 8-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and/or ⁇ 4-desaturase are expressed under the control of their own, preferably a different promoter, since repeating sequence motifs can lead to instability of the T-DNA or to recombination events.
  • the expression cassette is advantageously constructed in such a way that a promoter is followed by a suitable interface for insertion of the nucleic acid to be expressed, and then, if necessary, a terminator is located behind the polylinker in a polylinker.
  • This sequence is repeated several times, preferably three, four or five times, so that up to five genes are brought together in a construct and can thus be introduced into the transgenic plant for expression.
  • the sequence is advantageously repeated up to three times.
  • the nucleic acid sequences are inserted behind the promoter via the appropriate interface, for example in the polylinker.
  • Each nucleic acid sequence advantageously has its own promoter and, if necessary, its own terminator.
  • Such advantageous constructs are, for example, in DE 10102337 or DE 10102338 disclosed.
  • nucleic acid sequences behind a promoter and, if necessary, in front of a terminator.
  • the insertion site or the sequence of the inserted nucleic acids in the expression cassette is not of crucial importance, that is, a nucleic acid sequence can be inserted at the first or last position in the cassette without this significantly influencing the expression.
  • Different promoters such as the USP, LegB4 or DC3 promoter and different terminators can advantageously be used in the expression cassette.
  • the transcription of the introduced genes should advantageously be terminated by suitable terminators at the 3' end of the introduced biosynthesis genes (behind the stop codon).
  • suitable terminators at the 3' end of the introduced biosynthesis genes (behind the stop codon).
  • the OCS1 Terminator can be used here.
  • different terminator sequences should be used for each gene.
  • the gene construct can also include other genes that are to be introduced into the organisms. It is possible and advantageous to introduce and express regulatory genes, such as genes for inducers, repressors or enzymes, which intervene in the regulation of one or more genes of a biosynthetic pathway through their enzyme activity, into the host organisms. These genes can be of heterologous or homologous origin. Furthermore, further biosynthesis genes of fatty acid or lipid metabolism can advantageously be contained in the nucleic acid construct or gene construct, or these genes can be located on another or several further nucleic acid constructs.
  • nucleic acid sequences are biosynthesis genes of fatty acid or lipid metabolism selected from the group of acyl-CoA:lysophospholipid acyltransferase, ⁇ 3-desaturase, ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 8-desatuase, ⁇ 9-desaturase, ⁇ 12-desaturase , ⁇ 5-elongase, ⁇ 6-elongase and/or ⁇ 9-elongase.
  • acyl-CoA:lysophospholipid acyltransferase ⁇ 3-desaturase, ⁇ 4-desaturase, ⁇ 5-desaturase, ⁇ 6-desaturase, ⁇ 8-desatuase, ⁇ 9-desaturase, ⁇ 12-desaturase , ⁇ 5-elongase, ⁇ 6-elongase and/or ⁇
  • nucleic acids or genes can be cloned in combination with other elongases and desaturases in expression cassettes, such as those mentioned above, and used to transform plants with the help of Agrobacterium.
  • the regulatory sequences or factors can preferably positively influence and thereby increase the gene expression of the introduced genes.
  • the regulatory elements can thus advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and/or enhancers.
  • the expression cassettes can be used directly for introduction into the plant or can be introduced into vectors.
  • These advantageous vectors contain the nucleic acids used in the process, which are responsible for the ⁇ 12-desaturases, ⁇ 3-desaturases, ⁇ 9-elongases, ⁇ 6-desaturases, ⁇ 8-desaturases, ⁇ 6-elongases, ⁇ 5-desaturases, ⁇ 5-elongases or ⁇ 4 -Desaturases, or a nucleic acid construct, which contains the nucleic acid used alone or in combination with other biosynthetic genes of fatty acid or lipid metabolism such as the acyl-CoA:lysophospholipid acyltransferases, ⁇ 3-desaturases, ⁇ 4-desaturases, ⁇ 5-desaturases, ⁇ 6-desaturases, ⁇ 8-desatuases, ⁇ 9-desaturases, ⁇ 12-desaturases, ⁇ 3-desaturases, ⁇ 5-elongases, ⁇ 6-elongases and/or ⁇ 9-e
  • vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is bound.
  • plasmid which is a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome.
  • Certain vectors can replicate autonomously in a host cell into which they have been introduced (e.g. bacterial vectors with a bacterial origin of replication). Other vectors are advantageously integrated into the genome of a host cell when introduced into the host cell and are thereby replicated together with the host genome.
  • certain vectors can control the expression of genes to which they are operably linked.
  • vectors are referred to herein as “expression vectors”.
  • expression vectors suitable for recombinant DNA techniques take the form of plasmids.
  • plasmid and “vector” may be used interchangeably since plasmid is the most commonly used form of vector.
  • the invention is intended to encompass these other expression vector forms, such as viral vectors, which perform similar functions.
  • vector should also include other vectors known to those skilled in the art, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA.
  • the recombinant expression vectors advantageously used in the method comprise the nucleic acids described below or the gene construct described above in a form which is suitable for expressing the nucleic acids used in a host cell, which means that the recombinant expression vectors contain one or more regulatory sequences, selected on the basis of host cells to be used for expression, which are operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in such a way that expression of the nucleotide sequence is possible and they are linked to one another so that both sequences fulfill the predicted function assigned to the sequence (e.g.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g. polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990 ), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Florida, Ed.: Glick and Thompson, Chapter 7, 89-108 , including the references therein.
  • Regulatory sequences include those that control the constitutive expression of a nucleotide sequence in many host cell types and those that control the direct expression of the nucleotide sequence only in certain host cells under certain conditions.
  • the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the level of expression of the desired protein, etc.
  • the recombinant expression vectors used can be used to express ⁇ 12-desaturases, ⁇ 3-desaturases, ⁇ 9-elongases, ⁇ 6-desaturases, ⁇ 8-desaturases, ⁇ 6-elongases, ⁇ 5-desaturases, ⁇ 5-elongases and/or ⁇ 4-desaturases in prokaryotic or eukaryotic cells be designed. This is advantageous because intermediate vector construction steps are often carried out in microorganisms for simplicity.
  • Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990 ).
  • the recombinant expression vector can alternatively be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, DB, and Johnson, KS (1988) Gene 67:31-40 ), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), in which Gtutathione S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
  • GST Gtutathione S-transferase
  • Suitable inducible non-fusion E. coli expression vectors include pTrc ( Amann et al. (1988) Gene 69:301-315 ) and pET 11d ( Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89 ).
  • Target gene expression from the pTrc vector relies on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 11 d vector is based on transcription from a T7-gn10-lac fusion promoter, which is mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is provided by host strains BL21 (DE3) or HMS 174 (DE3) by a resident ⁇ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
  • vectors suitable in prokaryotic organisms are known to those skilled in the art; these vectors are, for example, in E. coli pLG338, pACYC184, the pBR series, such as pBR322, the pUC series, such as pUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, ⁇ gt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or p AJ667.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in the yeast S. cerevisiae include pYe-Desaturasec1 ( Baldari et al. (1987) Embo J 6:229-234 ), pMFa ( Kurjan and Herskowitz (1982) Cell 30:933-943 ), pJRY88 ( Schultz et al. (1987) Gene 54:113-123 ) and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Vectors and methods for constructing vectors suitable for use in other fungi, such as the filamentous fungi include those described in detail in: van den Hondel, CAMJJ, & Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, JF Peberdy et al., eds., pp. 1-28, Cambridge University Press: Cambridge , or in: More Gene Manipulations in Fungi [JW Bennet & LL Lasure, Eds., pp. 396-428: Academic Press: San Diego ].
  • Other suitable yeast vectors are, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.
  • the ⁇ 12-desaturases, ⁇ 3-desaturases, ⁇ 9-elongases, ⁇ 6-desaturases, ⁇ 8-desaturases, ⁇ 6-elongases, ⁇ 5-desaturases, ⁇ 5-elongases and/or ⁇ 4-desaturases can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series ( Smith et al. (1983) Mol. Cell Biol. 3:2156-2165 ) and the pVL series ( Lucklow and Summers (1989) Virology 170:31-39 ).
  • plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R.
  • a plant expression cassette preferably contains regulatory sequences that can control gene expression in plant cells and are operably linked so that each sequence can fulfill its function, such as termination of transcription, for example polyadenylation signals.
  • Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as gene 3 of the Ti plasmid pTiACH5, known as octopine synthase ( Gielen et al., EMBO J. 3 (1984) 835ff .) or functional equivalents thereof, but also all other terminators that are functionally active in plants are suitable.
  • a plant expression cassette preferably contains other operably linked sequences, such as translation enhancers, for example the overdrive sequence, which contains the 5'-untranslated leader sequence from tobacco mosaic virus, which contains the protein/RNA ratio increased, contains ( Gallie et al., 1987, Nucl. Acid Research 15:8693-8711 ).
  • the plant gene to be experimented must be operably linked to a suitable promoter as described above, which carries out gene expression in a timely, cell or tissue-specific manner.
  • suitable promoters are constitutive promoters ( Benfey et al., EMBO J. 8 (1989) 2195-2202 ), such as those derived from plant viruses, such as 35S CAMV ( Franck et al., Cell 21 (1980) 285-294 ), 19S CaMV (see also US 5352605 and WO 84/02913 ) or plant promoters, such as the one in US 4,962,028 described the small subunit of Rubisco.
  • telomeres are preferred sequences for use for functional association in plant gene expression cassettes.
  • targeting sequences that are necessary for directing the gene product into its appropriate cellular compartment (see a review in Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited therein), for example in the vacuole, the cell nucleus, all types of plastids such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • plastids such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes and other compartments of plant cells.
  • Plant gene expression can also be facilitated via a chemically inducible promoter as described above (see a review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108 ).
  • Chemically inducible promoters are particularly suitable when it is desired that gene expression occurs in a time-specific manner. Examples of such promoters include a salicylic acid-inducible promoter ( WO 95/19443 ), a tetracycline-inducible promoter ( Gatz et al. (1992) Plant J. 2, 397-404 ) and an ethanol-inducible promoter.
  • Promoters that respond to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRP1 gene promoter ( Ward et al., Plant. Mol. Biol. 22 (1993) 361-366 ), the heat-inducible hsp80 promoter from tomato ( US 5,187,267 ), the cold-inducible alpha-amylase promoter from potato ( WO 96/12814 ) or the wound-inducible pinII promoter ( EP-A-0 375 091 ).
  • the pathogen-induced PRP1 gene promoter Ward et al., Plant. Mol. Biol. 22 (1993) 361-366
  • the heat-inducible hsp80 promoter from tomato US 5,187,267
  • the cold-inducible alpha-amylase promoter from potato
  • WO 96/12814 the wound-inducible pinII promoter
  • promoters are preferred which bring about gene expression in tissues and organs in which fatty acid, lipid and oil biosynthesis takes place, in sperm cells, such as the cells of the endosperm and the developing embryo.
  • Suitable promoters are the napingen promoter from rapeseed ( US 5,608,152 ), the USP promoter from Vicia faba ( Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67 ), the oleosin promoter from Arabidopsis ( WO 98/45461 ), the phaseolin promoter from Phaseolus vulgaris ( US 5,504,200 ), the Bce4 promoter from Brassica ( WO 91/13980 ) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9 ) as well as promoters that bring about seed-specific expression in monocotyledonous plants such as corn, barley, wheat, rye,
  • Suitable promoters of note are the Ipt2 or Ipt1 gene promoter from barley ( WO 95/15389 and WO 95/23230 ) or the in WO 99/16890 described (promoters from the barley hordein gene, the rice glutelin gene, the rice orycin gene, the rice prolamin gene, the wheat gliadin gene, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene).
  • the multiparallel expression of the ⁇ 12-desaturases, ⁇ 3-desaturases, ⁇ 9-elongases, ⁇ 6-desaturases, ⁇ 8-desaturases, ⁇ 6-elongases, ⁇ 5-desaturases, ⁇ 5-elongases and / or ⁇ 4-desaturases used in the process may be desired.
  • Such expression cassettes can be introduced via simultaneous transformation of several individual expression constructs or preferably by combining several expression cassettes on one construct.
  • Several vectors, each with several expression cassettes can also be transformed and transferred to the host cell.
  • Promoters that bring about plastid-specific expression are also particularly suitable, since plastids are the compartment in which the precursors and some end products of lipid biosynthesis are synthesized.
  • Suitable promoters such as the viral RNA polymerase promoter, are described in WO 95/16783 and WO 97/06250 , and the clpP promoter from Arabidopsis described in WO 99/46394 .
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection”, conjugation and transduction, as used herein, are intended to cover a variety of methods known in the art for introducing foreign nucleic acid (e.g. DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE- Dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment.
  • Suitable methods for transforming or transfecting host cells, including plant cells, can be found in Sambrook et al.
  • Host cells that are in principle suitable for taking up the nucleic acid, the gene product or the vector are all prokaryotic or eukaryotic organisms.
  • the host organisms advantageously used are microorganisms, such as fungi or yeasts or plant cells, preferably plants or parts thereof.
  • Mushrooms, yeasts or plants are preferably used, particularly preferably plants, very particularly preferably plants, such as oil crop plants, which contain large amounts of lipid compounds, such as rapeseed, evening primrose, hemp, thistle, peanut, canola, linseed, soy, safflower, sunflower, borage , or plants such as corn, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, marigolds, solanaceous plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bush plants ( coffee, cocoa, tea), Salix species, trees (oil palm, coconut) as well as perennial grasses and forage crops.
  • Particularly preferred plants according to the invention are oil fruit plants, such as soy, peanut, rapeseed, canola, linseed, hemp, evening primrose, sunflower, safflower, trees (oil palm, coconut).
  • nucleic acids with a sequence which code for polypeptides with ⁇ 5 elongase activity the elongase encoded by the nucleic acid sequences being C 16 and C 18 fatty acids with a double bond and advantageously polyunsaturated C 18 fatty acids with a ⁇ 6 Double bond and polyunsaturated C 20 fatty acids reacted with a ⁇ 5 double bond.
  • C 22 fatty acids are not elongated.
  • Disclosed isolated nucleic acids are represented by nucleic acid sequences which encode polypeptides with ⁇ 5-elongase activity and which contain an amino acid sequence selected from the group of an amino acid sequence with the in SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141 or SEQ ID NO: 142 shown sequence.
  • Preferred nucleic acid sequences that code for polypeptides with ⁇ 5-elongase activity advantageously contain the aforementioned amino acid sequences. These are described in more detail in Table 2.
  • Nucleic acid sequences that code for ⁇ 6-elongases, ⁇ 3-desaturases, ⁇ 6-desaturases, ⁇ 5-desaturases, ⁇ 4-desaturases or ⁇ 12-desaturases are listed below.
  • nucleic acids come from organisms such as non-human animals, ciliates, fungi, plants such as algae or dinoflagellates that can synthesize PUFAs.
  • the isolated above-mentioned nucleic acid sequences come from the order Salmoniformes,
  • isolated nucleic acids with sequences that code for polypeptides with ⁇ 3-desaturase activity the ⁇ 3-desaturases encoded by the nucleic acid sequences being C 18 , C 20 and C 22 fatty acids with two, three, four or five double bonds and advantageously polyunsaturated C 18 fatty acids with two or three double bonds and polyunsaturated C 20 fatty acids with two, three or four double bonds.
  • C 22 fatty acids with four or five double bonds are also desaturated.
  • isolated nucleic acids with sequences that code for polypeptides with ⁇ 12-desaturase activity ⁇ 4-desaturases, ⁇ 5-desaturases and ⁇ 6-desaturases
  • nucleic acid (molecule) also includes the untranslated sequence located at the 3' and 5' ends of the coding gene region: at least 500, preferably 200, particularly preferably 100 nucleotides of the sequence upstream the 5' end of the coding region and at least 100, preferably 50, particularly preferably 20 nucleotides of the sequence downstream of the 3' end of the coding gene region.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid preferably has no sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (e.g., sequences located at the 5' and 3' ends of the nucleic acid).
  • the isolated ⁇ 5-elongase molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally represent the nucleic acid molecule in genomic DNA flank the cell from which the nucleic acid comes.
  • nucleic acid molecules used in the method for example a nucleic acid molecule with a nucleotide sequence of SEQ ID NO: 67 or part thereof, can be isolated using standard molecular biology techniques and the sequence information provided here. Comparison algorithms can also be used to identify, for example, a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level. These can be used as a hybridization probe as well as using standard hybridization techniques (such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 ) can be used to isolate further nucleic acid sequences that are useful in the process.
  • a nucleic acid molecule comprising a complete sequence of SEQ ID NO: 67 or a part thereof can be isolated by polymerase chain reaction using oligonucleotide primers based on this sequence or parts thereof (e.g. a nucleic acid molecule comprising the complete sequence or a part of it, can be isolated by polymerase chain reaction using oligonucleotide primers created on the basis of this same sequence).
  • mRNA can be isolated from cells (e.g. by the guanidinium thiocyanate extraction method of Chirgwin et al.
  • oligonucleotide primers for polymerase chain reaction amplification can be created based on one of the sequences shown in SEQ ID NO: 67 or using the amino acid sequences shown in SEQ ID NO: 68.
  • a nucleic acid can be amplified using cDNA or alternatively genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • nucleic acid amplified in this way can be cloned into a suitable vector and characterized using DNA sequence analysis.
  • Oligonucleotides corresponding to a desaturase nucleotide sequence can be prepared by standard synthetic methods, for example with an automated DNA synthesizer.
  • Homologues of the ⁇ 5-elongase nucleic acid sequences used with the sequence SEQ ID NO: 67 mean, for example, allelic variants with at least about 50 or 60%, preferably at least about 60 or 70%, more preferably at least about 70 or 80%, 90% or 95% and even more preferably at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity or homology to a nucleotide sequence shown in SEQ ID NO: 67 or its homologues, derivatives or analogues or parts thereof.
  • the entire sequence can also be used advantageously.
  • Allelic variants include in particular functional variants that can be obtained by deletion, insertion or substitution of nucleotides from/in the sequence shown in SEQ ID NO: 67, but the intention is that the enzyme activity of the synthesized proteins resulting therefrom is necessary for the insertion of a or more genes is advantageously retained and wherein the nucleic acid does not have the sequence shown in SEQ ID No: 113 and wherein the amino acid sequence does not have the amino acid sequence shown in SEQ ID NO: 114.
  • Proteins that still have the enzymatic activity of the ⁇ 5-elongase, that is, whose activity is essentially not reduced means proteins with at least 10%, preferably 20%, particularly preferably 30%, very particularly preferably 40% of the original enzyme activity, compared to the protein encoded by SEQ ID NO: 67.
  • SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO : 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 , SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 41, SEQ
  • the homology was calculated over the entire amino acid or nucleic acid sequence range.
  • a number of programs based on different algorithms are available to those skilled in the art for comparing different sequences.
  • the algorithms from Needleman and Wunsch or Smith and Waterman deliver particularly reliable results.
  • the program PileUp was used for the sequence comparisons ( J. Mol. Evolution., 25, 351-360, 1987 , Higgins et al., CABIOS, 5 1989: 151-153 ) or the programs Gap and BestFit [ Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970 ) and Smith and Waterman (Adv. Appl. Math.
  • SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77
  • SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77
  • the promoters upstream of the specified nucleotide sequences can be modified by one or more nucleotide exchanges, by insertion(s) and/or deletion(s), without however disturbing the functionality or activity of the promoters becomes. It is also possible that the activity of the promoters is increased by modification of their sequence or that they are completely replaced by more active promoters, even from heterologous organisms.
  • nucleic acids and protein molecules with ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 9-elongase, ⁇ 6-desaturase, ⁇ 8-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and/or ⁇ 4- Desaturase activity which is involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds across membranes, is advantageously used in the method according to the invention for modulating the production of PUFAs in transgenic organisms in plants such as corn, wheat, rye , oats, triticale, rice, barley, soybean, peanut, cotton, Linum species such as oil or fiber flax, Brassica species such as rapeseed, canola and turnip, pepper, sunflower, borage, evening primrose and marigold, solanacaene plants such as potato , tobacco, eggplant and
  • PUFAs polyunsaturated fatty acids
  • Brasicaceae, Boraginaceae, Primulaceae, or Linaceae are particularly suitable for the production of PUFAs, for example stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid.
  • Flax Linum usitatissimum is particularly advantageous for producing PUFAS with the nucleic acid sequences according to the invention, as described, in combination with other desaturases and elongases.
  • Lipid synthesis can be divided into two parts: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate and the addition or modification of a polar headgroup.
  • Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phosphoglycerides.
  • Fatty acid synthesis begins with the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase or to acetyl-ACP by acetyltransacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions to yield a saturated fatty acid molecule with the desired chain length.
  • acyl-CoA:lysophospholipid acyltransferases Furthermore, these enzymes can transfer the elongated fatty acids from the CoA esters to the phospholipids. If necessary, this reaction sequence can be repeated several times.
  • Precursors for PUFA biosynthesis are, for example, oleic acid, linoleic and linolenic acid. These C18 carbon fatty acids must be extended to C20 and C22 to obtain eicosa and docosa chain type fatty acids.
  • arachidonic acid eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid
  • arachidonic acid eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid
  • arachidonic acid eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid
  • eicosapentaenoic acid and /or docosahexaenoic acid can advantageously be used to produce eicosapentaenoic acid and /or docosahexaenoic acid and then used for various purposes in food, feed, cosmetic or pharmaceutical applications.
  • C 20 and/or C 22 fatty acids with at least two, advantageously at least three, four, five or six double bonds in the fatty acid molecule preferably C 20 or C 22 fatty acids with advantageously four, five or six double bonds in the fatty acid molecule getting produced.
  • the desaturation can take place before or after elongation of the corresponding fatty acid.
  • the products of desaturase activities and possible further desaturation and elongation lead to preferred PUFAs with higher degrees of desaturation, including further elongation of C 20 to C 22 fatty acids, to fatty acids such as ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, stearidonic acid, Eicosatetraenoic acid or eicosapentaenoic acid.
  • Substrates of the desaturases and elongases used in the process according to the invention are C 16 , C 18 or C 20 fatty acids such as, for example, linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, eicosatetraenoic acid or stearidonic acid.
  • Preferred substrates are linoleic acid, ⁇ -linolenic acid and/or ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
  • the synthesized C 20 or C 22 fatty acids with at least two, three, four, five or six double bonds in the fatty acid are obtained in the process according to the invention in the form of the free fatty acid or in the form of their esters, for example in the form of their glycerides.
  • glycolide is understood to mean a glycerol esterified with one, two or three carboxylic acid residues (mono-, di- or triglyceride). “Glyceride” is also understood to mean a mixture of different glycerides. The glyceride or the glyceride mixture can contain other additives, for example free fatty acids, antioxidants, proteins, carbohydrates, vitamins and/or other substances.
  • a “glyceride” in the sense of the process according to the invention also means derivatives derived from glycerol.
  • these also include glycerophospholipids and glyceroglycolipids.
  • the glycerophospholipids such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholipids are preferred as examples.
  • fatty acids must then be transported to various modification sites and incorporated into the triacylglycerol storage lipid.
  • Another important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol fatty acid acyltransferase (see Frentzen, 1998, Lipid, 100(4-5):161-166 ).
  • the PUFAs produced in the process include a group of molecules that higher animals can no longer synthesize and therefore have to absorb, or which higher animals can no longer produce sufficiently themselves and therefore have to absorb additionally, even though they are easily synthesized by other organisms, such as bacteria for example, cats can no longer synthesize arachidonic acid.
  • Phospholipids in the context of the invention are to be understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and/or phosphatidylinositol, advantageously phosphatidylcholine.
  • the terms production or productivity are well known in the art and include the concentration of the fermentation product (compounds of formula I) formed in a certain time period and a certain fermentation volume (e.g. kg of product per hour per liter). It also includes the productivity within a plant cell or a plant, that is, the content of the desired fatty acids produced in the process based on the content of all fatty acids in this cell or plant.
  • production efficiency encompasses the time required to achieve a certain production quantity (e.g. how long it takes the cell to achieve a certain throughput rate of a fine chemical).
  • yield or product/carbon yield is well known in the art and encompasses the efficiency of converting the carbon source into the product (i.e. the fine chemical). This is usually expressed, for example, as kg of product per kg of carbon source.
  • yield or production of the compound By increasing the yield or production of the compound, the amount of recovered molecules or appropriate recovered molecules of that compound in a given amount of culture is increased over a set period of time.
  • biosynthesis or biosynthetic pathway are well known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step and highly regulated process.
  • degradation or degradation pathway are well known in the art and encompass the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), for example in a multi-step and highly regulated process.
  • metabolism is well known in the art and encompasses the entirety of biochemical reactions that take place in an organism.
  • the metabolism of a specific compound e.g. the metabolism of a fatty acid
  • derivatives of the nucleic acid molecule are represented in SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63 , SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO : 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:
  • the homology was calculated over the entire amino acid or nucleic acid sequence range.
  • the program PileUp was used for the sequence comparisons ( J. Mol. Evolution., 25, 351-360, 1987 , Higgins et al., CABIOS, 5 1989: 151-153 ) or the programs Gap and BestFit [ Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970 ) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981 )], which are included in the GCG software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)].
  • sequence homology values given above in percent were determined using the BestFit program over the entire sequence range with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10,000 and Average Mismatch: 0.000. Unless otherwise stated, the default settings were always used for sequence comparisons.
  • the disclosure also includes nucleic acid molecules that are different from any of those in SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 131
  • SEQ ID NO: 43 SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 131, SEQ ID NO: 133 or SEQ ID NO: 183 shown ⁇ 12-
  • Nucleic acid molecules advantageous for alternative methods can be based on their homology to the ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 5-elongase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase and/or ⁇ 6-elongase disclosed herein -Nucleic acids are isolated using the sequences or part thereof as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • isolated nucleic acid molecules can be used that are at least 15 nucleotides long and, under stringent conditions, with the nucleic acid molecules that have a nucleotide sequence of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID
  • Nucleic acids with at least 25, 50, 100, 250 or more nucleotides can also be used.
  • hybridized under stringent conditions as used herein is intended to describe hybridization and washing conditions under which nucleotide sequences that are at least 60% homologous to one another usually remain hybridized to one another.
  • the conditions are preferably such that sequences are at least about 65%, more preferably at least about 70% and more preferably at least about 75% or more are homologous to one another, usually remaining hybridized to one another.
  • These stringent conditions are known to those skilled in the art and can be summarized in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6 ., find.
  • SSC sodium chloride/sodium citrate
  • these hybridization conditions differ depending on the type of nucleic acid and, for example, if organic solvents are present, with regard to the temperature and concentration of the buffer.
  • the temperature differs between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 ⁇ SSC (pH 7.2).
  • the temperature under standard conditions is about 42°C.
  • the hybridization conditions for DNA:DNA hybrids are, for example, 0.1 x SSC and 20°C to 45°C, preferably between 30°C and 45°C.
  • the hybridization conditions for DNA:RNA hybrids are, for example, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C.
  • Gaps can be inserted into the sequence of a protein or nucleic acid to create an optimal alignment with the other protein or nucleic acid).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding site in the other sequence, then the molecules at that position are homologous (i.e., amino acid or nucleic acid "homology,” as used herein, corresponds to amino acid - or nucleic acid “identity”).
  • the programs or algorithms used are described above.
  • Mutations can occur in one of the sequences of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO : 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 131, SEQ ID NO: 133 or SEQ ID NO
  • conservative amino acid substitutions are made at one or more of the predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” involves swapping the amino acid residue for an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • non-polar side chains e.g alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g. threonine, valine, isoleucine
  • aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine.
  • a predicted non-essential amino acid residue in a ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase or ⁇ 6-elongase is thus preferably replaced by another amino acid residue from the same side chain family.
  • the mutations may be introduced randomly across all or part of the ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase or ⁇ 6-elongase coding sequence, for example by Saturation mutagenesis, and the resulting mutants can be screened for the ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase or ⁇ 6-elongase activity described herein to To identify mutants that retained ⁇ 12-desaturase, ⁇ 3-desaturase, ⁇ 6-desaturase, ⁇ 5-desaturase, ⁇ 4-desaturase, ⁇ 5-elongase, or ⁇ 6-elongase activity.
  • transgenic non-human organisms which have the nucleic acids SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 131, SEQ ID NO: 133 or
  • the sequencing of recombinant DNA molecules was carried out using a laser fluorescence DNA sequencer from ABI using the Sanger method ( Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74, 5463-5467 ). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
  • RNA of Oncoryhnchus mykiss was isolated using the RNAeasy kit from Qiagen (Valencia, CA, US). Poly-A+ RNA (mRNA) was isolated from the total RNA using oligo-dT cellulose (Sambrook et al., 1989). The RNA was reverse transcribed using the Promega Reverse Transcription System Kit and the synthesized cDNA was cloned into the lambda ZAP vector (lambda ZAP Gold, Stratagene). According to the manufacturer's instructions, the cDNA was unpacked into plasmid DNA. The cDNA plasmid library was then used for PCR to clone expression plasmids.
  • the PCR product was incubated for 2 h at 37°C with the restriction enzymes HindIII and BamHI.
  • the yeast expression vector pYES3 (Invitrogen) was incubated in the same way.
  • the 812 bp or 905 bp PCR product and the vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA is purified using the Qiagen Gel purification kit according to the manufacturer's instructions. Vector and Elongase cDNA were then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • pYES3-OmELO2 and pYES3-OmELO3 were verified by sequencing and transformed into the Saccharomyces strain INVSc1 (Invitrogen) by electroporation (1500 V).
  • pYES3 was transformed in parallel.
  • the yeasts were then plated on complete minimal medium without tryptophan with 2% glucose. Cells that were capable of growth without tryptophan in the medium therefore contain the corresponding plasmids pYES3, pYES3-OmELO2 (SEQ ID NO: 51) and pYES3-OmELO3 (SEQ ID NO: 53). After selection, two transformants were selected for further functional expression.
  • the PCR products were incubated for 16 h at 37°C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP was incubated in the same way.
  • the PCR products and the 7624 bp vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA is purified using the Qiagen Gel purification kit according to the manufacturer's instructions.
  • the vector and PCR products were then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmids pSUN-OmELO2 and pSUN-OmELO3 were verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP ( Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ).
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the octopine synthase gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 )
  • the USP promoter corresponds to nucleotides 1-684 (Genbank Accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was using a commercially available T7 standard primer (Stratagene) and amplified using a synthesized primer via a PCR reaction according to standard methods. (Primer sequence: 5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3', SEQ ID NO: 174).
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
  • Example 6 Lipid extraction from yeasts and seeds:
  • the effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound can be determined by growing the modified microorganisms or the modified plant under appropriate conditions (such as those described above) and that Medium and/or the cellular components are examined for the increased production of the desired product (ie lipids or a fatty acid).
  • a desired compound such as a fatty acid
  • These analysis techniques are known to those skilled in the art and include spectroscopy, thin-layer chromatography, staining processes of various types, enzymatic and microbiological processes as well as analytical chromatography, such as high-performance liquid chromatography (see for example Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.
  • plant lipids are obtained from plant material such as Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22):12935-12940 , and Browse et al. (1986) Analytic Biochemistry 152:141-145 , described extracted.
  • the qualitative and quantitative lipid or fatty acid analysis is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2 ); Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: Oily Press, 1989 , Repr. 1992, IX, 307 p. (Oily Press Lipid Library; 1 ); " Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) - 16 (1977 ) udT: Progress in the Chemistry of Fats and Other Lipids CODEN.
  • the analytical procedures include measurements of nutrient amounts in the medium (e.g. sugars, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of biomass composition and growth, analysis of the production of common metabolites from biosynthetic pathways, and measurements of gases produced during fermentation. Standard procedures for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF Stanbury, eds., IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773 ) and the references cited therein are described.
  • medium e.g. sugars, hydrocarbons, nitrogen sources, phosphate and other ions
  • fatty acids abbreviations: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatography-mass spectrometry; TAG, triacylglycerol; TLC, thin layer chromatography.
  • the material to be analyzed can be broken down by ultrasonication, glass milling, liquid nitrogen and milling, or other applicable methods.
  • the material must be centrifuged after breaking.
  • the sediment is dissolved in distilled water. resuspended, heated at 100°C for 10 min, cooled on ice and centrifuged again, followed by extraction in 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 h at 90°C, resulting in hydrolyzed oil and lipid compounds, which result in transmethylated lipids.
  • fatty acid methyl esters are extracted into petroleum ether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 microm, 0.32 mm) at a temperature gradient between 170°C and 240°C Subjected to 240°C for 20 min and 5 min.
  • the identity of the fatty acid methyl esters obtained must be defined using standards available from commercial sources (i.e. Sigma).
  • Plant material is first homogenized mechanically using mortars to make it more amenable to extraction.
  • the cell sediment is hydrolyzed with 1 M methanolic sulfuric acid and 2% dimethoxypropane for 1 h at 90 ° C and the lipids are transmethylated.
  • the resulting fatty acid methyl esters (FAME) are extracted into petroleum ether.
  • the extracted FAME are analyzed by gas-liquid chromatography with a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature gradient from 170 ° C to 240 ° C in 20 min and 5 min at 240 °C analyzed.
  • the identity of the fatty acid methyl esters is confirmed by comparison with corresponding FAME standards (Sigma).
  • the identity and position of the double bond can be further analyzed by appropriate chemical derivatization of the FAME mixtures, for example to 4,4-dimethoxyoxazoline derivatives (Christie, 1998) using GC-MS.
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • OmELO2 shows no elongase activity, while OmELO3 has been shown to have significant activity with various substrates.
  • the substrate specificity of OmElo3 could be determined after expression and feeding of various fatty acids ( Figure 2 ).
  • the fed substrates can be detected in large quantities in all transgenic yeasts. All transgenic yeasts show the synthesis of new fatty acids, the products of the OmElo3 reaction. This means that the OmElo3 gene could be functionally expressed.
  • FIG 2 shows that the OmElo3 has a substrate specificity that leads to the elongation of ⁇ 5 and ⁇ 6 fatty acids with a ⁇ 3 double bond with high specificity. Furthermore, ⁇ 6 fatty acids (C 18 and C 20 ) could also be elongated with less specificity. Stearidonic acid (C 18:4 ⁇ 3) and eicosapentaenoic acid (C 20:5 ⁇ 3) represent the best substrates for the OmElo3 (up to 66% elongation).
  • the reconstitution of the biosynthesis of DHA was started from EPA (C 20:5 w3) or stearidonic acid (C 18:4 ⁇ 3) by co-expression of OmElo3 with the ⁇ 4-desaturase from Euglena gracilis or the ⁇ 5-desaturase from Phaeodactylum tricornutum and the ⁇ 4-desaturase from Euglena gracilis .
  • the expression vectors pYes2-EgD4 and pESCLeu-PtD5 were constructed.
  • the yeast strain mentioned above which was already transformed with pYes3-OmElo3 (SEQ ID NO: 55), was further transformed with pYes2-EgD4 or simultaneously with pYes2-EgD4 and pESCLeu-PtD5.
  • the transformed yeasts were selected on complete minimal medium agar plates with 2% glucose, but without tryptophan and uracil in the case of the pYes3-OmELO/pYes2-EgD4 strain and without tryptophan, uracil and leucine in the case of the pYes3-OmELO/pYes2- EgD4+pESCLeu-PtD5 strain. Expression was induced by the addition of 2% (w/v) galactose as stated above. The cultures were incubated for a further 120 h at 15°C.
  • Figure 3 shows the fatty acid profiles of transgenic yeasts fed with C 20:5 ⁇ 3.
  • C 20:5 ⁇ 3 was elongated very efficiently to C 22:5 ⁇ 3 (65% elongation).
  • the additional introduction of the Eg ⁇ 4 desaturase led to the conversion of C 22:5 ⁇ 3 to C 22:6 ⁇ 3 (DHA).
  • the fatty acid composition of the transgenic yeasts is in Figure 5 reproduced. After co-expression of OmElo3 and EgD4, up to 3% DHA could be detected in yeast.
  • OmElo3, EgD4 and PtD5 sequences used in this invention are suitable for the production of DHA in eukaryotic cells.
  • Petioles or hypocotyledons of freshly germinated sterile rapeseed plants are incubated in a Petri dish with a 1:50 agrobacteria dilution for 5-10 minutes. This is followed by a 3-day colncubation in the dark at 25°C on 3MS medium with 0.8% Bacto agar. The cultivation is continued after 3 days with 16 hours of light/8 hours of darkness and at a weekly rhythm on MS medium with 500 mg/l Claforan (cefotaxime sodium), 50 mg/l kanamycin, 20 microM benzylaminopurine (BAP) and 1, 6 g/l glucose continued. Growing shoots are transferred to MS medium with 2% sucrose, 250 mg/L Claforan and 0.8% Bacto agar. If no roots form after three weeks, 2-indolebutyric acid was added to the medium as growth hormone for rooting.
  • 2-indolebutyric acid was added to the medium as growth hormone for rooting.
  • Regenerated shoots are maintained on 2MS medium with kanamycin and claforan, transferred to soil after rooting and, after cultivation for two weeks in a climate chamber or greenhouse, grown, flowered, mature seeds harvested and tested for elongase expression such as ⁇ 5-elongase or ⁇ 6-elongase activity or ⁇ 3-desaturase activity was examined using lipid analyses. Lines with increased levels of C 20 and C 22 polyunsaturated fatty acids can be identified in this way.
  • transgenic flax plants can, for example, be carried out using the method of Bell et al., 1999, In Vitro Cell. Dev. Biol.-Plant. 35(6):456-465 be generated using particle bombardment.
  • an Agrobacterium-mediated transformation was, for example, after Mlynarova et al. (1994), Plant Cell Report 13: 282-285 used for linen transformation.
  • Example 10 Cloning of ⁇ 5-Elonaase genes from Thraustochytrium aureum ATCC34304 and Thraustochytrium ssp.
  • RNA of T. aureum ATCC34304 and Thraustochytrium ssp. was isolated using the RNAeasy kit from Qiagen (Valencia, CA, US). mRNA was isolated from the total RNA using the PolyATract isolation system (Promega). The mRNA was reverse transcribed using the Marathon cDNA Amplification Kit (BD Biosciences) and adapters were ligated according to the manufacturer's instructions. The cDNA library was then used for PCR to clone expression plasmids using 5' and 3' RACE (rapid amplification of cDNA ends).
  • PCR products BioTaurELO1 see SEQ ID NO: 65
  • TL16y2 see SEQ ID NO: 83
  • the PCR product is ligated into the vector by a T overhang and the activity of a topoisomerase (Invitrogen).
  • E. coli DH5 ⁇ cells were transformed.
  • Corresponding clones were identified by PCR, the plasmid DNA was isolated using the Qiagen DNAeasy kit and verified by sequencing. The correct sequence was then transformed into Saccharomyces strain INVSc1 (Invitrogen) by electroporation (1500 V).
  • the empty vector pYES2.1 was transformed in parallel.
  • the yeasts were then plated on complete minimal medium without uracil with 2% glucose. Cells that were able to grow in the medium without uracil therefore contain the corresponding plasmids pYES2.1, pYES2.1-BioTaurELO1 and pYES2.1-TL16y2. After selection, two transformants were selected for further functional expression.
  • the PCR products were incubated for 16 h at 37°C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP was incubated in the same way.
  • the PCR products and the 7624 bp vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA is purified using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • the vector and PCR products were then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmids pSUN-BioTaurELO1 and pSUN-TL16y2 were verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP ( Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ).
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the octopine synthase gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 )
  • the USP promoter corresponds to nucleotides 1-684 (Genbank Accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
  • the substrate specificity of BioTaurELO1 could be determined after expression and feeding of various fatty acids ( Figure 6).
  • 200 ⁇ M ⁇ -linolenic acid and eicosapentaenoic acid were added to the yeast incubation medium and incubated for 24 h.
  • the fatty acids were extracted from the yeasts, they were transmethylated and separated by gas chromatography.
  • the elongation products formed from the two fed fatty acids are marked by arrows.
  • the fed substrates can be detected in large quantities in all transgenic yeasts. All transgenic yeasts show the synthesis of new fatty acids, the products of the BioTaurELO1 reaction. This means that the BioTaurELO1 gene could be functionally expressed.
  • FIG. 6 shows that the BioTaurELO1 has a substrate specificity that leads to the elongation of ⁇ 5 and ⁇ 6 fatty acids with a ⁇ 3 double bond with high specificity.
  • ⁇ 6 fatty acids C 18 and C 20
  • ⁇ -linolenic acid C 18:3 ⁇ 6 with 65.28%
  • stearidonic acid C 18:4 ⁇ 3
  • eicosapentaenoic acid C 20:5 ⁇ 3 with 22.01% conversion are converted.
  • the substrate specificities of the different feeding experiments are shown in Table 3 (see at the end of the description).
  • the conversion rate of GLA when fed GLA and EPA was 65.28%.
  • the conversion rate of EPA with the same feeding of GLA and EPA was 9.99%.
  • the conversion rate of EPA was 22.01%.
  • the conversion rate was 14.47%.
  • TL16y2 The functionality and substrate specificity of TL16y2 could vary according to expression and feeding Fatty acids can be determined.
  • Table 4 shows the feeding experiments. The feeding experiments were carried out in the same way as described for BioTaurELO1. The fed substrates can be detected in large quantities in all transgenic yeasts. The transgenic yeasts showed the synthesis of new fatty acids, the products of the TL16y2 reaction ( Figure 11 ). This means that the TL16y2 gene could be functionally expressed.
  • Table 4 Expression of TL16y2 in yeast.
  • Table 4 show the following percentage conversions with TL16y2 compared to the control: a) % conversion EPA (250 ⁇ M): 8%, b) % conversion EPA (50 ⁇ M): 41%, c) % conversion ARA: 20, 3%, d) % sales SDA: 79, 4% and e) % sales GLA: 74.9%.
  • TL16y2 therefore shows ⁇ 5, ⁇ 6 and ⁇ 8 elongase activity.
  • the activity is highest for C 18 fatty acids with a ⁇ 6 double bond.
  • C 20 fatty acids with a ⁇ 5 or ⁇ 8 double bond are then elongated.
  • OtElo1 shows the highest similarity to an elongase from Danio rerio (GenBank AAN77156; ca. 26% identity), while OtElo2 has the greatest similarity to Physcomitrella Elo (PSE) [ca. 36% identity] (alignments were performed using the tBLASTn algorithm ( Altschul et al., J. Mol. Biol. 1990, 215: 403 - 410 ) carried out.
  • the Saccharomyces cerevisiae strain 334 was transformed by electroporation (1500 V) with the vector pOTE1 or pOTE2.
  • a yeast transformed with the empty vector pYES2 was used as a control.
  • the transformed yeasts were selected on complete minimal medium (CMdum) agar plates with 2% glucose but without uracil. After selection, three transformants were selected for further functional expression.
  • precultures each consisting of 5 ml of CMdum liquid medium with 2% (w/v) raffinose but without uracil were first inoculated with the selected transformants and incubated for 2 days at 30 ° C, 200 rpm.
  • 5 ml of CMdum liquid medium (without uracil) with 2% raffinose and 300 ⁇ M various fatty acids were then inoculated with the precultures to an OD 600 of 0.05.
  • Expression was induced by the addition of 2% (w/v) galactose. The cultures were incubated for a further 96 h at 20°C.
  • the PCR products were incubated for 16 h at 37°C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP was incubated in the same way.
  • the PCR products and the vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA was purified using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • the vector and PCR products were then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmids pSUN-OtELO1 and pSUN-OtELO2 were verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP ( Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ).
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the Ostreococcus gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 ).
  • the USP promoter corresponds to nucleotides 1 to 684 (Genbank accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8):761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • the substrate specificity of OtElo1 could be determined after expression and feeding of various fatty acids (Table 5).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtElo1 reaction. This means that the OtElo1 gene could be functionally expressed.
  • OtElo1 has narrow substrate specificity.
  • the OtElo1 could only produce the C 20 fatty acids eicosapentaenoic acid ( Figure 7 ) and arachidonic acid ( Figure 8 ), but preferred the ⁇ 3-desaturated eicosapentaenoic acid.
  • Table 5 shows the substrate specificity of the elongase OtElo1 for C 20 polyunsaturated fatty acids with a double bond in the ⁇ 5 position towards various fatty acids.
  • the yeasts that had been transformed with the vector pOTE1 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the substrate specificity of OtElo2 (SEQ ID NO: 81) could be determined after expression and feeding of various fatty acids (Table 6).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtElo2 reaction. This means that the OtElo2 gene could be functionally expressed.
  • Table 6 shows the substrate specificity of the elongase OtElo2 towards various fatty acids.
  • the yeasts that had been transformed with the vector pOTE2 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the corresponding primer pairs were selected to reflect the yeast consensus sequence for highly efficient translation ( Kozak, Cell 1986, 44:283-292 ) next to the start codon.
  • the amplification of the TpElo DNAs was carried out with 1 ⁇ L cDNA, 200 ⁇ M dNTPs, 2.5 U Advantage polymerase and 100 pmol of each primer in a total volume of 50 ⁇ l.
  • the conditions for PCR were as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 1 minute and 72°C for 2 minutes, and a final extension step at 72 °C for 10 minutes.
  • the PCR products were incubated for 30 min at 21°C with the yeast expression vector - pYES2.1-TOPO (Invitrogen) according to the manufacturer's instructions.
  • the PCR product is ligated into the vector by a T overhang and the activity of a topoisomerase (Invitrogen).
  • E. coli DH5 ⁇ cells were transformed.
  • Corresponding clones were identified by PCR, the plasmid DNA was isolated using the Qiagen DNAeasy kit and verified by sequencing. The correct sequence was then transformed into Saccharomyces strain INVSc1 (Invitrogen) by electroporation (1500 V). As a control, the empty vector pYES2.1 was transformed in parallel.
  • the yeasts were then plated on complete minimal medium without uracil with 2% glucose. Cells without uracil in the Medium were capable of growth and therefore contained the corresponding plasmids pYES2.1, pYES2.1-TpELO1, pYES2.1-TpELO2 and pYES2.1-TpELO3. After selection, two transformants were selected for further functional expression.
  • the PCR products are incubated with the restriction enzyme Notl for 16 hours at 37°C.
  • the plant expression vector pSUN300-USP is incubated in the same way.
  • the PCR products and the 7624 bp vector are then separated by agarose gel electrophoresis and the corresponding DNA fragments are cut out.
  • the DNA is purified using the Qiagen Gel Purification Kit according to the manufacturer’s instructions.
  • the vector and PCR products are then ligated.
  • the Rapid Ligation Kit from Roche is used for this purpose.
  • the resulting plasmids pSUN-TPELO1, pSUN-TPELO2 and pSUN-TPELO3 are verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP ( Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ).
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the octopine synthase gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 )
  • the USP promoter corresponds to nucleotides 1-684 (Genbank Accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and linseed.
  • Example 22 Expression of TpEL01, TpELO2 and TpELO3 in yeast
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8):761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • the substrate specificity of TpElo1 could be determined after expression and feeding of various fatty acids ( Figure 9 ).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the TpElo1 reaction. This means that the TpElo1 gene could be functionally expressed.
  • TpElo1 has narrow substrate specificity. TpElo1 could only elongate the C 20 fatty acids eicosapentaenoic acid and arachidonic acid, but preferred the ⁇ 3-desaturated eicosapentaenoic acid.
  • the yeasts transformed with the vector pYES2-TpELO1 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC.
  • Table 7 Expression of TpELO1 in yeast.
  • columns 1 and 3 are the control reactions for columns 2 (fed 250 ⁇ M C ⁇ sub>20:4 ⁇ /sub> ⁇ 5,8,11,14) and 4 (fed 250 ⁇ M C ⁇ sub>20:5 ⁇ /sub> ⁇ 5,8,11,14,17).
  • the substrate specificity of TpElo3 could be determined after expression and feeding of various fatty acids ( Figure 10 ).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the TpElo3 reaction. This means that the TpElo3 gene could be functionally expressed.
  • Table 8 shows that the TpElo3 has a narrow substrate specificity.
  • the TpElo3 could only elongate the C 18 fatty acids ⁇ -linolenic acid and stearidonic acid, but preferred the ⁇ 3-desaturated stearidonic acid.
  • the yeasts transformed with the vector pYES2-TpELO3 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC.
  • Table 8 Expression of TpELO3 in yeast. Column 1 shows the fatty acid profile of yeast without feeding. Column 2 shows the control reaction. In columns 3 to 6, ⁇ -linolenic acid, stearidonic acid, arachidonic acid, and eicosapentaenoic acid were fed (250 ⁇ M of each fatty acid).
  • Example 24 Cloning of an expression plasmid for the heterologous expression of Pi-omega3Des in yeast
  • the PCR product was incubated for 2 h at 37°C with the restriction enzymes Hindlil and BamHI.
  • the yeast expression vector pYES3 (Invitrogen) was incubated in the same way.
  • the 1104 bp PCR product and the vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA is purified using the Qiagen Gel purification kit according to the manufacturer's instructions. Vector and desaturase cDNA were then ligated. The Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmid pYES3-Pi-omega3Des was checked by sequencing and transformed into the Saccharomyces strain INVSc1 (Invitrogen) by electroporation (1500 V). As a control, pYES3 was transformed in parallel. The yeasts were then plated on complete minimal medium without tryptophan with 2% glucose. Cells that were able to grow without tryptophan in the medium therefore contain the corresponding plasmids pYES3, pYES3-Pi-omega3Des. After selection, two transformants were selected for further functional expression.
  • Example 25 Cloning of expression plasmids for seed-specific expression in plants
  • the PCR products were incubated for 4 h at 37°C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP was incubated in the same way.
  • the PCR products and the 7624 bp vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA is purified using the Qiagen Gel purification kit according to the manufacturer's instructions.
  • the vector and PCR products were then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmid pSUN-Pi-omega3Des was verified by sequencing.
  • the substrate specificity of Pi-omega3Des could be determined after expression and feeding of various fatty acids ( Figures 12 to 18 ).
  • the fed substrates are present in large quantities in all transgenic yeasts, which proves the uptake of these fatty acids by the yeasts.
  • the transgenic yeasts demonstrate the synthesis of new fatty acids, the products of the Pi-omega3Des reaction. This means that the Pi-omega3Des gene could be functionally expressed.
  • Figure 12 represents the desaturation of linoleic acid (C 18:2 ⁇ 6 fatty acid) to ⁇ -linolenic acid (C 18:3 ⁇ 3 fatty acid) by the Pi-omega3Des.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells using the empty vector pYES2 ( Figure 12 A) or the vector pYes3-Pi-omega3Des ( Figure 12 B) had been transformed.
  • the yeasts were cultured in minimal medium in the presence of C 18:2 ⁇ 9,12 fatty acid (300 ⁇ M).
  • the FAMEs were then analyzed via GLC.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells using the empty vector pYES2 ( Figure 13 A) or the vector pYes3-Pi-omega3Des ( Figure 13 B) had been transformed.
  • the yeasts were cultured in minimal medium in the presence of ⁇ -C 18:3 ⁇ 6,9,12 fatty acid (300 ⁇ M).
  • the FAMEs were then analyzed via GLC.
  • Figure 14 represents the desaturation of C 20:2 ⁇ 6 fatty acid to C 20:3 ⁇ 3 fatty acid by Pi-omega3Des.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells that had been transformed with the empty vector pYES2 (FIG. 14 A) or the vector pYes3-Pi-omega3Des (FIG. 14 B).
  • the yeasts were cultured in minimal medium in the presence of C 20:2 ⁇ 11,14 fatty acid (300 ⁇ M).
  • the FAMEs were then analyzed via GLC.
  • Figure 15 represents the desaturation of C 20:3 ⁇ 6 fatty acid to C 20:4 ⁇ 3 fatty acid by Pi-omega3Des.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells using the empty vector pYES2 ( Figure 15 A) or the vector pYes3-Pi-omega3Des ( Figure 15 B) had been transformed.
  • the yeasts were cultured in minimal medium in the presence of C 20:3 ⁇ 8,11,14 fatty acid (300 ⁇ M). The FAMEs were then analyzed via GLC.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells using the empty vector pYES2 ( Figure 16 A) or the vector pYes3-Pi-omega3Des ( Figure 16 B) had been transformed.
  • the yeasts were cultured in minimal medium in the presence of C 20:4 ⁇ 5,8,11,14 fatty acid (300 ⁇ M).
  • the FAMEs were then analyzed via GLC.
  • Figure 17 represents the desaturation of docosatetraenoic acid (C 22:4 ⁇ 6 fatty acid) to docosapentaenoic acid (C 22:5 ⁇ 3 fatty acid) by Pi-omega3Des.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells using the empty vector pYES2 ( Figure 17 A) or the vector pYes3-Pi-omega3Des ( Figure 17 B) had been transformed.
  • the yeasts were cultured in minimal medium in the presence of C 22:4 ⁇ 7,10,13,16 fatty acid (300 ⁇ M).
  • the FAMEs were then analyzed via GLC.
  • the substrate specificity of Pi-omega3Des towards different fatty acids is Figure 18 refer to.
  • the yeasts transformed with the vector pYes3-Pi-omega3Des were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC. Each value represents an average of three measurements.
  • the conversion rates (% desaturation) were calculated using the formula: [product]/[product]+[substrate]*100.
  • Pi-omega3Des can also be used to produce transgenic plants. Lipid extraction can then be carried out from the seeds of these plants as described in Example 6.
  • Example 28 Cloning of desaturase genes from Ostreococcus tauri
  • the cloning was done as follows: 40 ml of an Ostreococcus tauri culture in the stationary phase were centrifuged and resuspended in 100 ⁇ l bidest water and stored at -20°C. Based on the PCR method, the associated genomic DNAs were amplified. The corresponding primer pairs were selected to reflect the yeast consensus sequence for highly efficient translation ( Kozak, Cell 1986, 44:283-292 ) next to the start codon. Amplification of the OtDes DNAs was performed with 1 ⁇ L of thawed cells, 200 ⁇ M dNTPs, 2.5 U of Taq polymerase, and 100 pmol of each primer in a total volume of 50 ⁇ L. The conditions for PCR were as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 1 minute and 72°C for 2 minutes, and a final extension step at 72 °C for 10 minutes.
  • Saccharomyces cerevisiae strain 334 was transformed with the vector pYES2.1-OtDes6.1 by electroporation (1500 V).
  • a yeast transformed with the empty vector pYES2 was used as a control.
  • the transformed yeasts were selected on complete minimal medium (CMdum) agar plates with 2% glucose but without uracil. After selection, three transformants were selected for further functional expression.
  • CMdum liquid medium For the expression of the OtDes6.1 desaturase, precultures each consisting of 5 ml of CMdum liquid medium with 2% (w/v) raffinose but without uracil were first inoculated with the selected transformants and incubated for 2 days at 30 ° C, 200 rpm. 5 ml of CMdum liquid medium (without uracil) with 2% raffinose and 300 ⁇ M various fatty acids were then inoculated with the precultures to an OD 600 of 0.05. Expression was induced by the addition of 2% (w/v) galactose. The cultures were incubated for a further 96 h at 20°C.
  • the PCR products are incubated with the restriction enzyme Notl for 16 hours at 37°C.
  • the plant expression vector pSUN300-USP is incubated in the same way.
  • the PCR products and the vector are then separated using agarose gel electrophoresis and the corresponding DNA fragments are cut out.
  • the DNA is purified using the Qiagen Gel Purification Kit according to the manufacturer’s instructions.
  • the vector and PCR products are then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmids are verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP ( Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ).
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the Ostreococcus gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 ).
  • the USP promoter corresponds to nucleotides 1 to 684 (Genbank accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8):761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • the substrate specificity of desaturases can be determined after expression in yeast (see examples cloning of desaturase genes, yeast expression) by feeding with different yeasts. Descriptions for determining the individual activities can be found in WO 93/11245 for ⁇ 15 desaturases, WO 94/11516 for ⁇ 12-desaturases, WO 93/06712 , US 5,614,393 , US 5614393 , WO 96/21022 , WO 0021557 and WO 99/27111 for ⁇ 6-desaturases, Qiu et al. 2001, J. Biol. Chem. 276, 31561-31566 for ⁇ 4-desaturases, Hong et al. 2002, Lipids 37,863-868 for ⁇ 5-desaturases.
  • Table 9 shows the substrate specificity of the desaturase OtDes6.1 towards various fatty acids.
  • the substrate specificity of OtDes6.1 could be determined after expression and feeding of various fatty acids.
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtDes6.2 reaction ( Figure 20 ). This means that the OtDes6.1 gene could be functionally expressed.
  • the yeasts that had been transformed with the vector pYES2-OtDes6.1 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the activity corresponds to the conversion rate calculated according to [substrate/(substrate+product)*100].
  • Table 9 shows that OtDes6.1 has a substrate specificity for linoleic and linolenic acid (C ⁇ sub>18:2 ⁇ /sub> and C ⁇ sub>18:3 ⁇ /sub>), as the highest activities with these fatty acids can be achieved.
  • Figure 20 shows the conversion of linoleic acid by OtDes6.1.
  • the FAMEs were analyzed using gas chromatography.
  • the fed substrate (C 18:2 ) is converted to ⁇ -C 18:3 . Both the starting material and the resulting product are marked by arrows.
  • Figure 21 clearly shows that the reaction products GLA and STA of the ⁇ 6-desaturase OtDes6.1 are elongated almost quantitatively to DHGLA and DHSTA, respectively, in the presence of the ⁇ 6-elongase PSE1.
  • the subsequent desaturation by the ⁇ 5-desaturase PtD5 also occurs smoothly to form ARA or EPA.
  • Approximately 25 to 30% of the elongase product is desaturated ( Figure 21 B and D ).
  • the open reading frame of the respective DNA is cloned downstream of the galactose-inducible GAL1 promoter from pYES2.1/V5-His-TOPO (Invitrogen), obtaining the corresponding pYES2.1 clones.
  • the Saccharomyces cerevisiae strain 334 is transformed by electroporation (1500 V) with the vectors pYES2.1-TpDesaturases.
  • a yeast transformed with the empty vector pYES2 is used as a control.
  • the transformed yeasts are selected on complete minimal medium (CMdum) agar plates with 2% glucose but without uracil. After selection, three transformants are selected for further functional expression.
  • precultures each consisting of 5 ml of CMdum liquid medium with 2% (w/v) raffinose but without uracil are first inoculated with the selected transformants and incubated for 2 days at 30 ° C, 200 rpm.
  • 5 ml of CMdum liquid medium (without uracil) with 2% raffinose and 300 ⁇ M various fatty acids are then inoculated with the precultures to an OD 600 of 0.05.
  • Expression is induced by the addition of 2% (w/v) galactose.
  • the cultures are incubated for a further 96 h at 20°C.
  • the PCR products are incubated with the restriction enzyme Notl for 16 hours at 37°C.
  • the plant expression vector pSUN300-USP is incubated in the same way.
  • the PCR products and the vector are then separated using agarose gel electrophoresis and the corresponding DNA fragments are cut out.
  • the DNA is purified using the Qiagen Gel Purification Kit according to the manufacturer’s instructions.
  • the vector and PCR products are then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmids are verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP ( Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ).
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the OCS gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 ).
  • the USP promoter corresponds to nucleotides 1 to 684 (Genbank accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and linseed.
  • the signals are identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8):761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • the substrate specificity of desaturases can be determined after expression in yeast (see examples cloning of desaturase genes, yeast expression) by feeding with different yeasts. Descriptions for determining the individual activities can be found in WO 93/11245 for ⁇ 15 desaturases, WO 94/11516 for ⁇ 12-desaturases, WO 93/06712 , US 5,614,393 , WO 96/21022 , WO 0021557 and WO 99/27111 for ⁇ 6-desaturases, Qiu et al. 2001, J. Biol. Chem. 276, 31561-31566 for ⁇ 4-desaturases, Hong et al. 2002, Lipids 37,863-868 for ⁇ 5-desaturases.
  • the activity of the individual desaturases is calculated from the conversion rate using the formula [substrate/(substrate+product)*100].
  • Tables 11 and 12 provide an overview of the cloned Thalassiosira pseudonana desaturases.
  • Table 11 Length and characteristic features of the cloned Thalassiosira desaturases.
  • TpD4 2633 496-1314 1571-2260 Thrautochitrium D4-des 56%/43% TpD5-1 2630 490-800 900-2019 Phaeodactylum D5-des 74%/62% TpD5-2 2643 532-765 854-2068 Phaeodactylum D5-des 72%/61% TpD6 2371 379-480 630-1982 Phaeodactylum D6-des 83%/69% TpFAD2 2667 728-2032 - Phaeodactylum FAD2 76% / 61% TpO3 2402 403-988 1073-1743 Chaenorhabdidis Fad2 49%/28%
  • the ⁇ 12-desaturase genes from Ostreococcus and Thalassiosira can also be cloned.
  • Example 38 Cloning of Elongase genes from Xenopus laevis and Ciona intestinalis
  • the cDNA clone of X. laevis was obtained from NIH (National Institute of Health) [ Genetic and genomic tools for Xenopus research: The NIH Xenopus initiative, Dev. Dyn. 225 (4), 384-391 (2002 )].
  • the cDNA clone of C. inetstinalis was obtained from the University of Kyto [ Satou,Y., Yamada,L., Mochizuki,Y., Takatori,N., Kawashima,T., Sasaki,A., Hamaguchi,M., Awazu,S., Yagi,K., Sasakura,Y., Nakayama,A., Ishikawa,H., Inaba,K. and Satoh,N. "A cDNA resource from the basal chordate Ciona intestinalis" JOURNAL Genesis 33 (4), 153-154 (2002 )].
  • the amplification of the Elongase DNAs was carried out with 1 ⁇ L cDNA, 200 ⁇ M dNTPs, 2.5 U Advantage polymerase and 100 pmol of each primer in a total volume of 50 ⁇ l.
  • the conditions for PCR were as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 1 minute and 72°C for 2 minutes, and a final extension step at 72 °C for 10 minutes.
  • the PCR products were incubated for 30 min at 21 °C with the yeast expression vector - pYES2.1-TOPO (Invitrogen) according to the manufacturer's instructions.
  • the PCR product is ligated into the vector using a T overhang and the activity of a topoisomerase (Invitrogen) according to the manufacturer's instructions.
  • E. coli DH5 ⁇ cells were transformed.
  • Corresponding clones were identified by PCR, the plasmid DNA was isolated using the Qiagen DNAeasy kit and verified by sequencing. The correct sequence was then transformed into Saccharomyces strain INVSc1 (Invitrogen) by electroporation (1500 V). As a control, the empty vector pYES2.1 was transformed in parallel.
  • yeasts were then plated on complete minimal medium without uracil with 2% glucose. Cells that were capable of growth in the medium without uracil therefore contain the corresponding plasmids pYES2.1, pYES2.1-ELO(XI) and pYES2.1-ELO(Ci). After selection, two transformants were selected for further functional expression.
  • Example 40 Cloning of expression plasmids for seed-specific expression in plants
  • the PCR products were incubated for 16 h at 37°C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP was incubated in the same way.
  • the PCR products and the 7624 bp vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA was purified using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • the vector and PCR products were then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmids pSUN-ELO(XI) and pSUN-ELO(Ci) were verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP [ Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ].
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the octopine synthase gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 )
  • the USP promoter corresponds to nucleotides 1-684 (Genbank accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • Primer sequence 5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3' (SEQ ID NO: 129).
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8): 761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • the substrate specificity of ELO(XI) could be determined after expression and feeding of various fatty acids ( Figure 22 ).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the ELO(XI) reaction. This means that the ELO(XI) gene could be functionally expressed.
  • Table 13 shows that the ELO(XI) has broad substrate specificity. Both C 18 and C 20 fatty acids are elongated, with a preference for ⁇ 5 and ⁇ 6 desaturated fatty acids being observed.
  • the yeasts transformed with the vector pYES2-ELO(XI) were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC.
  • Table 13 Expression of ELO(XI) in yeast. The conversion rate of various educts (fed 250 ⁇ M each) is described.
  • the substrate specificity of ELO(Ci) could be determined after expression and feeding of various fatty acids ( Figure 23 ).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the ELO (Ci) reaction. This means that the ELO(Ci) gene could be functionally expressed.
  • Table 14 Expression of ELO(Ci) in yeast. The conversion rate of various educts (fed 250 ⁇ M each) is described.
  • Table 14 shows that the ELO(Ci) has a broad substrate specificity. Both C 18 and C 20 fatty acids are elongated, with a preference for ⁇ 5 and ⁇ 6 desaturated fatty acids being observed.
  • the yeasts that had been transformed with the vector pYES2-ELO(Ci) were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC.
  • OtElo1 and OtElo1.2 show the highest similarity to an elongase from Danio rerio (GenBank AAN77156; ca. 26% identity), while OtElo2 and OtElo2.1 have the greatest similarity to Physcomitrella Elo (PSE) [ca. 36% identity] (alignments were performed using the tBLASTn algorithm ( Altschul et al., J. Mol. Biol. 1990, 215: 403 - 410 ) carried out.
  • the open reading frames of the respective DNAs were cloned downstream of the galactose-inducible GAL1 promoter from pYES2.1/V5-His-TOPO (Invitrogen), where pOTE1, pOTE1.2, pOTE2 and pOTE2. 1 were received.
  • the Saccharomyces cerevisiae strain 334 was transformed by electroporation (1500 V) with the vector pOTE1, pOTE1.2, pOTE2 and pOTE2.1, respectively.
  • a yeast transformed with the empty vector pYES2 was used as a control.
  • the transformed yeasts were selected on complete minimal medium (CMdum) agar plates with 2% glucose but without uracil. After selection, three transformants were selected for further functional expression.
  • precultures each consisting of 5 ml of CMdum liquid medium with 2% (w/v) raffinose but without uracil were first inoculated with the selected transformants and incubated for 2 days at 30 ° C, 200 rpm.
  • 5 ml of CMdum liquid medium (without uracil) with 2% raffinose and 300 ⁇ M various fatty acids were then inoculated with the precultures to an OD 600 of 0.05.
  • Expression was induced by the addition of 2% (w/v) galactose. The cultures were incubated for a further 96 h at 20°C.
  • the PCR products are incubated with the restriction enzyme Notl for 16 hours at 37°C.
  • the plant expression vector pSUN300-USP is incubated in the same way.
  • the PCR products and the vector were then separated by agarose gel electrophoresis and the corresponding DNA fragments were cut out.
  • the DNA was purified using the Qiagen Gel Purification Kit according to the manufacturer's instructions.
  • the vector and PCR products were then ligated.
  • the Rapid Ligation Kit from Roche was used for this purpose.
  • the resulting plasmids pSUN-OtELO1, pSUN-OtELO1.2, pSUN-OtELO2 and pSUN-OtELO2.2 were verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP [ Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ].
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is the OCS gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 ).
  • the USP promoter corresponds to nucleotides 1 to 684 (Genbank accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • Primer sequence 5'-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3').
  • SEQ ID NO: 130 The PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and flaxseed.
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8):761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • the substrate specificity of OtElo1 could be determined after expression and feeding of various fatty acids (Table 15).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtElo1 reaction. This means that the OtElo1 gene could be functionally expressed.
  • Table 15 shows that OtElo1 and OtElo1.2 have narrow substrate specificity. OtElo1 and OtElo1.2 could only produce the C 20 fatty acids eicosapentaenoic acid ( Figure 24A,24B ) and arachidonic acid ( Figures 25A, 25B ) elongate, but preferred the w3-desaturated eicosapentaenoic acid.
  • Table 15 shows the substrate specificity of the elongase OtElo1 and OtElo1.2 for C20 polyunsaturated fatty acids with a double bond in the ⁇ 5 position compared to various fatty acids.
  • the yeasts that had been transformed with the vector pOTE1 or pOTE1.2 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC.
  • the substrate specificity of the OtElo2 (SEQ ID NO: 81) OtElo2.1 (SEQ ID NO: 111) could be determined after expression and feeding of various fatty acids (Table 16).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the OtElo2 reaction. This means that the OtElo2 and OtElo2.1 genes could be functionally expressed.
  • Table 16 shows the substrate specificity of the elongase OtElo2 and OtElo2.1 towards various fatty acids. OtElo2.1 shows significantly higher activity.
  • the yeasts that had been transformed with the vector pOTE2 or pOTE2.1 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC.
  • OtElo2 or OtElo2.1 is a ⁇ 6 elongase.
  • Table 16 fatty acid substrate Sales (in %) OtElo2 Sales (in %)OtELO2.2 16-0 - - 16:1 ⁇ 9 - - 16:3 ⁇ 7,10,13 - - 18:0 - - 18:1 ⁇ 6 - - 18:1 ⁇ 9 - - 18:1 ⁇ 11 - - 18:2 ⁇ 9.12 - - 18:3 ⁇ 6,9,12 15.3 55.7 18:3 ⁇ 5,9,12 - - 18:4 ⁇ 6,9,12,15 21.1 70.4 2O-2 ⁇ 11.14 - - 20:3 ⁇ 8,11,14 - - 20:4 ⁇ 5,8,11,14 - - 20:5 ⁇ 5,8,11,14,17 - - 22:4 ⁇ 7,10,13,16 - - 22:
  • Figure 24 A - D shows the elongation of eicosapentaenoic acid by OtElo1 (B) and OtElo1.2 (D), respectively.
  • the controls (A, C) do not show the product of elongation (C 22:5 ⁇ 3).
  • Figure 25 A - D shows the elongation of arachidonic acid by OtElo1 (B) and OtElo1.2 (D), respectively.
  • the controls (A, C) do not show the product of elongation (C 22:4 ⁇ 6).
  • Example 48 Cloning of elongase genes from Euglena gracilis and Arabidopsis thaliana
  • sequences from Arabidopsis thaliana or Euglena gracilis with corresponding motifs could be identified in sequence databases (Genbank, Euglena EST Bank). These are the following sequences: Gene name SEQ ID amino acids EGY1019 (E. gracilis) SEQ ID NO: 131 262 EGY2019 (E. gracilis) SEQ ID NO: 133 262 At3g06460 (A. thaliana) SEQ ID NO: 135 298 At3g06470 (A. thaliana) SEQ ID NO: 137 278
  • RNA from a four-day Euglena culture was isolated using the RNAeasy kit from Qiagen (Valencia, CA, US). Poly-A+ RNA (mRNA) was isolated from the total RNA using oligo-dT cellulose (Sambrook et al., 1989). The RNA was reverse transcribed using the Promega Reverse Transcription System Kit and the synthesized cDNA was cloned into the lambda ZAP vector (lambda ZAP Gold, Stratagene). According to the manufacturer's instructions, the cDNA was unpacked into plasmid DNA and clones were sequenced for random sequencing. mRNA was isolated from the total RNA using the PolyATract isolation system (Promega).
  • the mRNA was reverse transcribed using the Marathon cDNA Amplification Kit (BD Biosciences) and the adapters were ligated according to the manufacturer's instructions.
  • the cDNA library was then used for PCR to clone expression plasmids using 5' and 3' RACE (rapid amplification of cDNA ends).
  • RNA from A . thaliana was processed according to Chrigwin et al., (1979). Leaves from 21-day-old plants were ground in liquid nitrogen, digestion buffer was added, and incubated for 15 min at 37°C. After centrifugation (10 min, 4°C, 12000xg), the RNA in the supernatant was precipitated with 0.02 volume of 3 M sodium acetate pH 5.0 and 0.75 volume of ethanol at -20°C for 5 h. After a further centrifugation step, the RNA was then taken up in 1 mL TES per g of starting material, extracted once with a volume of phenol-chloroform and once with a volume of chloroform, and the RNA was precipitated with 2.5 M LiCl.
  • RNA was resuspended in water. According to Sambrook et al. In 1989, the cDNA was synthesized and RT-PCR was performed with the derived primers. The PCR products were cloned into the vector pYES2.1-TOPO (Invitrogen) according to the manufacturer's instructions.
  • the Saccharomyces cerevisiae strain 334 was transformed by electroporation (1500 V) with the vector pAt60 or pAt70.
  • a yeast transformed with the empty vector pYES2.1 was used as a control.
  • the transformed yeasts were selected on complete minimal medium (CMdum) agar plates with 2% glucose but without uracil. After selection, three transformants were selected for further functional expression.
  • At-elongases For the expression of the At-elongases, precultures each consisting of 5 ml of CMdum liquid medium with 2% (w/v) raffinose but without uracil were first inoculated with the selected transformants and incubated for 2 days at 30 ° C, 200 rpm.
  • CMdum liquid medium without uracil
  • various fatty acids were then inoculated with the precultures to an OD 600 of 0.05.
  • Expression was induced by the addition of 2% (w/v) galactose.
  • the cultures were incubated for a further 96 h at 20°C.
  • Example 50 Expression of pAt60 and pAt70 in yeast
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8):761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • the substrate specificity of the elongases At3g06460 and At3g06470 could be determined after expression and feeding of various fatty acids (Table 17, Figure 26 ).
  • the fed substrates can be detected in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the At3g06460 and At3g06470 genes, respectively. This means that these genes could be functionally expressed.
  • Table 17 Elongation of EPA by the elongases At3g06460 and At3g06470. Measurement of yeast extracts after feeding with 250 ⁇ M EPA.
  • At3g06460 gene Fed fatty acid Content ofC20:5n-3 Content of C22:5n-3 At3g06460 EPA (C20:5n-3) 8/20 0.6 At3g06460 EPA (C20:5n-3) 25.4 1.1 EPA conversion rate At3g06460: 3.0% At3g06470: 4.1%
  • Figure 26 represents the elongation of C 20: 5 n-3 by the elongation gases At3g06470.
  • Example 52 Cloning of an elongase from Phaeodactylum tricornutum
  • Nucleotide bases in brackets mean that there is a mixture of oligonucleotides, each with one or the other nucleotide base.
  • Frozen cells were ground to a fine powder after centrifugation in the presence of liquid nitrogen and mixed with 2 mL homogenization buffer (0.33 M sorbitol, 0.3 M NaCl, 10 mM EDTA, 10 mM EGTA, 2% SDS, 2% mercaptoethanol in 0.2 M Tris-CI pH 8.5) resuspended. After adding 4 mL of phenol and 2 mL of chloroform, the mixture was shaken vigorously at 40-50°C for 15 min. The mixture was then centrifuged (10 min x 10,000g) and the aqueous phase was gradually extracted with chloroform. Nucleic acids were then precipitated by adding 1/20 volume of 4 M sodium bicarbonate solution and centrifuged.
  • RNA pellet was taken up in 80 mM Tris-borate pH 7.0 and 1 mM EDTA and the RNA was precipitated with 8 M lithium chloride. After centrifugation and washing with 70% ethanol, the RNA pellet was taken up with RNAse-free water.
  • Poly(A) RNA was isolated using Dynabeads (Dynal, Oslo, Norway) according to the manufacturer's instructions, and first-strand cDNA synthesis was carried out using MLV-Rtase from Roche (Mannheim). The second-strand synthesis was then carried out using DNA polymerase I and Klenow fragment, followed by RnaseH digestion.
  • the cDNA was treated with T4 DNA polymerase and then attached to EcoRI/Xhol adapters (Pharmacia, Freiburg) using T4 ligase. After Xhol digestion, phosphorylation and gel separation, fragments larger than 300 bp were ligated into the lambda ZAP Express phage (Stratagene, Amsterdam, Netherlands) according to the manufacturer's instructions. After mass excision of the cDNA library and plasmid recovery, the plasmid library was transformed into E. coli DH10B cells and used for PCR screening.
  • the PCR fragment with the sequence number SEQ ID NO: 187 could be generated.
  • This fragment was labeled with digoxigenin (Roche, Mannheim) and used as a probe for screening the phage library.
  • sequence SEQ ID NO: 183 could be obtained, which represents the full-length RNA molecule of the ⁇ 6-elongase from Phaeodactylum:
  • the corresponding primer pairs were selected to reflect the yeast consensus sequence for highly efficient translation ( Kozak, Cell 1986, 44:283-292 ) next to the start codon.
  • the amplification of the PtELO6 DNA was carried out with 1 ⁇ L cDNA, 200 ⁇ M dNTPs, 2.5 U Advantage polymerase and 100 pmol of each primer in a total volume of 50 ⁇ L.
  • the conditions for PCR were as follows: initial denaturation at 95°C for 5 minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 1 minute and 72°C for 2 minutes, and a final extension step at 72 °C for 10 minutes.
  • the PCR products were incubated for 30 min at 21°C with the yeast expression vector - pYES2.1-TOPO (Invitrogen) according to the manufacturer's instructions.
  • the PCR product (see SEQ ID NO: 192) was ligated into the vector using a T overhang and the activity of a topoisomerase (Invitrogen). After incubation, E. coli DH5 ⁇ cells were transformed.
  • Corresponding clones were identified by PCR, the plasmid DNA was isolated using the Qiagen DNAeasy kit and verified by sequencing. The correct sequence was then transformed into Saccharomyces strain INVSc1 (Invitrogen) by electroporation (1500 V). As a control, the empty vector pYES2.1 was transformed in parallel.
  • the yeasts were then plated on complete minimal medium without uracil with 2% glucose. Cells that were able to grow in the medium without uracil therefore contain the corresponding plasmids pYES2.1 and pYES2.1-PtELO6. After selection, two transformants were selected for further functional expression.
  • Example 54 Cloning of expression plasmids for seed-specific expression in plants
  • the PCR products are incubated for 16 h at 37°C with the restriction enzyme Notl.
  • the plant expression vector pSUN300-USP is incubated in the same way.
  • the PCR products and the 7624 bp vector are then separated by agarose gel electrophoresis and the corresponding DNA fragments are cut out.
  • the DNA is purified using the Qiagen Gel Purification Kit according to the manufacturer’s instructions.
  • the vector and PCR products are then ligated.
  • the Rapid Ligation Kit from Roche is used for this purpose.
  • the resulting plasmid pSUN-PtELO is verified by sequencing.
  • pSUN300 is a derivative of the plasmid pPZP ( Hajdukiewicz, P, Svab, Z, Maliga, P., (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994 ).
  • pSUN-USP was created from pSUN300 by inserting a USP promoter into pSUN300 as an EcoRI fragment.
  • the polyadenylation signal is that of the octopine synthase gene from the A.
  • tumefaciens Ti plasmid (ocs terminator, Genbank accession V00088) ( De Greve, H., Dhaese, P., Seurinck, J., Lemmers, M., Van Montagu, M. and Schell, J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene J. Mol. Appl. Genet. 1 (6), 499-511 (1982 ).
  • the USP promoter corresponds to nucleotides 1-684 (Genbank accession X56240), with part of the non-coding region of the USP gene being contained in the promoter.
  • the 684 base pair promoter fragment was amplified using a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • a commercial T7 standard primer (Stratagene) and with the help of a synthesized primer via a PCR reaction according to standard methods.
  • the PCR fragment was cut with EcoRI/Sall and inserted into the vector pSUN300 with OCS terminator.
  • the plasmid named pSUN-USP was created. The construct was used to transform Arabidopsis thaliana, rapeseed, tobacco and linseed.
  • the signals were identified by comparing the retention times with corresponding fatty acid standards (Sigma).
  • the methodology is described, for example, in Napier and Michaelson, 2001, Lipids. 36(8):761-766 ; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585 , Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218 .
  • FIG 29 the conversion of C 18:3 ⁇ 6,9,12 and C 18:4 ⁇ 6,9,12,15 is shown.
  • the substrates are elongated by two carbon atoms each, producing the fatty acids C 20:3 ⁇ 8,11,14 and C 20:4 ⁇ 8,11,14,17 .
  • the substrate specificity of PtELO6 could be determined after expression and feeding of various fatty acids ( Figure 30 ).
  • the fed substrates can be detected in large quantities in all transgenic yeasts.
  • the transgenic yeasts showed the synthesis of new fatty acids, the products of the PtElo6 reaction. This means that the PtELO6 gene could be functionally expressed.
  • PtElo6 has a narrow substrate specificity.
  • PtELO6 could only elongate the C 18 fatty acids linoleic acid, linolenic acid, ⁇ -linolenic acid and stearidonic acid, but preferred the ⁇ 3-desaturated stearidonic acid (see also Figure 30 ).
  • the yeasts transformed with the vector pYES2-PtELO6 were cultured in minimal medium in the presence of the indicated fatty acids.
  • the fatty acid methyl esters were synthesized by acid methanolysis of intact cells.
  • the FAMEs were then analyzed via GLC. This is how the results were presented in the Figures 29 and 30 as well as those shown in Table 16.

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Claims (5)

  1. Procédé de fabrication d'acide docosahexénoïque (DHA) et d'acide eicosapenténoïque (EPA) dans des organismes non-humains transgéniques ayant une teneur d'au moins 1 % en poids en ces composés, par rapport à la teneur totale en lipides de l'organisme transgénique non-humain, caractérisé en ce qu'il comprend les étapes suivantes :
    a) introduction, dans l'organisme, d'au moins un acide nucléique ayant une séquence qui code pour une activité de Δ6-désaturase, et
    b) introduction, dans l'organisme, d'au moins un acide nucléique ayant une séquence qui code pour une activité de Δ6-élongase, et
    c) introduction, dans l'organisme, d'au moins un acide nucléique ayant une séquence qui code pour une activité de Δ5-désaturase, et
    d) introduction, dans l'organisme, d'au moins un acide nucléique ayant une séquence qui code pour une activité de Δ5-élongase, et
    e) introduction, dans l'organisme, d'un acide nucléique ayant une séquence qui code pour une activité de Δ4-désaturase, et
    l'acide nucléique ayant une séquence qui code pour le polypeptide ayant une activité de Δ5-élongase étant choisi dans le groupe consistant en:
    a) un acide nucléique ayant la séquence présentée dans SEQ ID NO:67, ou
    b) les acides nucléiques qui en conséquence du code génétique dégénéré peuvent dériver de la séquence d'acides aminés présentée dans SEQ ID NO:68, ou
    c) les dérivés de la séquence d'acide nucléique présentée dans SEQ ID NO:67, qui codent pour des polypeptides ayant une identité d'au moins 40 % au niveau des acides aminés avec SEQ ID NO:68, et présentent une activité de Δ5-élongase.
  2. Procédé selon la revendication 1, caractérisé en ce qu'on introduit en outre dans l'organisme un acide nucléique ayant une séquence qui code pour des polypeptides ayant une activité d'ω3-désaturase, choisi dans le groupe consistant en :
    a) un acide nucléique ayant la séquence présentée dans SEQ ID NO:87 ou SEQ ID NO:105, ou
    b) les acides nucléiques qui en conséquence du code génétique dégénéré peuvent dériver de la séquence d'acides aminés présentée dans SEQ ID NO:88 ou SEQ ID NO:106, ou
    c) les dérivés de la séquence d'acide nucléique présentée dans SEQ ID NO:87 ou SEQ ID N0:105, qui codent pour des polypeptides ayant une identité d'au moins 60 % au niveau des acides aminés avec SEQ ID NO:88 ou SEQ ID NO:106, et qui présentent une activité d'ω3-désaturase.
  3. Procédé selon les revendications 1 ou 2, caractérisé en ce qu'on introduit en outre dans l'organisme un acide nucléique ayant une séquence qui code pour des polypeptides ayant une activité de Δ12-désaturase, choisi dans le groupe consistant en :
    a) un acide nucléique ayant la séquence présentée dans SEQ ID NO:107 ou SEQ ID NO:109, ou
    b) les acides nucléiques qui en conséquence du code génétique dégénéré peuvent dériver de la séquence d'acides aminés présentée dans SEQ ID NO:108 ou SEQ ID NO:110, ou
    c) les dérivés de la séquence d'acide nucléique présentée dans SEQ ID NO:107 ou SEQ ID NO:109, qui codent pour des polypeptides ayant une identité d'au moins 60 % au niveau des acides aminés avec SEQ ID NO:108 ou SEQ ID NO:110 et qui présentent une activité de Δ12-désaturase.
  4. Procédé selon les revendications 1 à 2, caractérisé en ce que l'organisme transgénique non-humain est un microorganisme transgénique ou une plante transgénique.
  5. Procédé selon l'une des revendications 1 à 4, contenant l'étape de procédé supplémentaire de fabrication de produits pour l'alimentation humaine ou animale, de cosmétiques ou de produits pharmaceutiques contenant l'acide docosahexénoïque (DHA) et l'acide eicosapenténoïque (EPA)
EP09176083.5A 2003-08-01 2004-07-16 Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques Expired - Lifetime EP2166069B2 (fr)

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EP18155821.4A Withdrawn EP3395945A1 (fr) 2003-08-01 2004-07-16 Procédé de production de plusieurs acides gras insaturés dans des organismes transgeniques
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EP09176092.6A Revoked EP2169052B1 (fr) 2003-08-01 2004-07-16 Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques
EP09176085.0A Revoked EP2166089B1 (fr) 2003-08-01 2004-07-16 Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques
EP09176096.7A Expired - Lifetime EP2166090B1 (fr) 2003-08-01 2004-07-16 Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques
EP04763291.4A Expired - Lifetime EP1654344B1 (fr) 2003-08-01 2004-07-16 Procede de production d'acides gras polyinsatures dans des organismes transgeniques
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EP18155821.4A Withdrawn EP3395945A1 (fr) 2003-08-01 2004-07-16 Procédé de production de plusieurs acides gras insaturés dans des organismes transgeniques
EP09176088.4A Expired - Lifetime EP2172536B1 (fr) 2003-08-01 2004-07-16 Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques
EP09176076.9A Expired - Lifetime EP2166067B1 (fr) 2003-08-01 2004-07-16 Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques
EP09176092.6A Revoked EP2169052B1 (fr) 2003-08-01 2004-07-16 Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques
EP09176085.0A Revoked EP2166089B1 (fr) 2003-08-01 2004-07-16 Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques
EP09176096.7A Expired - Lifetime EP2166090B1 (fr) 2003-08-01 2004-07-16 Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques
EP04763291.4A Expired - Lifetime EP1654344B1 (fr) 2003-08-01 2004-07-16 Procede de production d'acides gras polyinsatures dans des organismes transgeniques
EP09176104.9A Expired - Lifetime EP2166071B1 (fr) 2003-08-01 2004-07-16 Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques
EP09176100.7A Expired - Lifetime EP2166070B1 (fr) 2003-08-01 2004-07-16 Procédé de production d'acides gras polyinsaturés chez des organismes transgéniques
EP09176093.4A Expired - Lifetime EP2169053B1 (fr) 2003-08-01 2004-07-16 Procédé de fabrication d'acides gras insaturés de manière multiple dans des organismes transgéniques

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Families Citing this family (79)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2713548T3 (es) 2003-08-01 2019-05-22 Basf Plant Science Gmbh Procedimiento para la producción de ácidos grasos poliinsaturados en organismos transgénicos
ATE430802T1 (de) 2003-08-21 2009-05-15 Monsanto Technology Llc Fettsäuredesaturasen aus primula
CA2556336C (fr) * 2004-02-17 2015-09-08 The University Of York Enzymes desaturases
EP2623584B1 (fr) * 2004-02-27 2019-04-10 BASF Plant Science GmbH Procédé de fabrication de plusieurs acides gras insaturés dans des plantes transgéniques
WO2005083053A2 (fr) * 2004-02-27 2005-09-09 Basf Plant Science Gmbh Procede pour produire des acides gras $g(v)-3 insatures dans des organismes transgeniques
BRPI0509944A (pt) 2004-04-16 2007-09-25 Monsanto Technology Llc expressão de dessaturases de ácido graxo em milho
CA3056110C (fr) * 2004-04-22 2020-07-14 Surinder Pal Singh Synthese d'acides gras polyinsatures a chaine longue par des cellules de recombinaison
DK1756280T3 (en) 2004-04-22 2015-02-02 Commw Scient Ind Res Org SYNTHESIS OF CHAIN, polyunsaturated fatty acids BY RECOMBINANT CELLS
US7550651B2 (en) 2004-06-25 2009-06-23 E.I. Du Pont De Nemours And Company Delta-8 desaturase and its use in making polyunsaturated fatty acids
JP4587451B2 (ja) * 2004-08-20 2010-11-24 サントリーホールディングス株式会社 ω３脂肪酸不飽和化活性を有するポリペプチドおよびそのポリペプチドをコードするポリヌクレオチドならびにそれらの利用
US7550286B2 (en) * 2004-11-04 2009-06-23 E. I. Du Pont De Nemours And Company Docosahexaenoic acid producing strains of Yarrowia lipolytica
DE102004063326A1 (de) * 2004-12-23 2006-07-06 Basf Plant Science Gmbh Verfahren zur Herstellung mehrfach ungesättigter Fettsäuren in transgenen Organismen
DE102005013779A1 (de) 2005-03-22 2006-09-28 Basf Plant Science Gmbh Verfahren zur Herstellung von mehrfach ungesättigten C20- und C22-Fettsäuren mit mindestens vier Doppelbindungen in transgenen Pflanzen
NZ563676A (en) * 2005-05-23 2011-01-28 Arcadia Biosciences Inc Safflower with elevated gamma-linolenic acid
DE102005038036A1 (de) * 2005-08-09 2007-02-15 Basf Plant Science Gmbh Verfahren zur Herstellung von Arachidonsäure und/oder Eicosapentaensäure in transgenen Nutzpflanzen
GB2431158A (en) 2005-10-13 2007-04-18 Rothamsted Res Ltd Process for the production of arachidonic and/or eicosapentaenoic acid
DK1951866T3 (da) 2005-11-23 2014-10-27 Du Pont Delta-9-elongaser og anvendelse heraf til fremstilling af flerumættede fedtsyrer
US7723574B2 (en) * 2005-11-24 2010-05-25 Basf Plant Science Gmbh Process for the production of Δ5-unsaturated fatty acids in transgenic organisms
JP4803584B2 (ja) * 2006-02-08 2011-10-26 独立行政法人産業技術総合研究所 脂質生産性の高い形質転換微生物
AR059376A1 (es) 2006-02-21 2008-03-26 Basf Plant Science Gmbh Procedimiento para la produccion de acidos grasos poliinsaturados
US7465793B2 (en) 2006-04-20 2008-12-16 E.I. Du Pont De Nemours And Company Synthetic Δ17 desaturase derived from Phytophthora ramourum and its use in making polyunsaturated fatty acids
US7695950B2 (en) * 2006-05-17 2010-04-13 E. I. Du Pont De Nemours And Company Δ5 desaturase and its use in making polyunsaturated fatty acids
US7678560B2 (en) * 2006-05-17 2010-03-16 E.I. Du Pont De Nemours And Company Δ 5 desaturase and its use in making polyunsaturated fatty acids
US9029111B2 (en) * 2006-08-24 2015-05-12 Basf Plant Science Gmbh Isolation and characterization of a novel pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains
AU2013202498B2 (en) * 2006-08-24 2015-04-30 Basf Plant Science Gmbh Isolation and characterization of a novel Pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains
CN101578363A (zh) 2006-08-29 2009-11-11 联邦科学技术研究组织 脂肪酸的合成
AU2013273704C1 (en) * 2006-10-06 2023-03-16 Basf Plant Science Gmbh Process for the production of polyunsaturated fatty acids in transgenic organisms
EP2182056B1 (fr) 2006-10-06 2015-12-23 BASF Plant Science GmbH Procédé de production d'acides gras polyinsaturés dans des organismes transgéniques non-humains
US20110016581A1 (en) 2006-10-23 2011-01-20 E.I. Du Pont De Nemours And Company Delta-8 desaturases and their use in making polyunsaturated fatty acids
US7709239B2 (en) 2006-12-07 2010-05-04 E.I. Du Pont De Nemours And Company Mutant Δ8 desaturase genes engineered by targeted mutagenesis and their use in making polyunsaturated fatty acids
WO2008073275A2 (fr) 2006-12-07 2008-06-19 E. I. Du Pont De Nemours And Company Gènes mutants de la désaturase delta 8 modifiés par mutagenèse ciblée et utilisation de ceux-ci dans la fabrication d'acides gras polyinsaturés
CA2673940C (fr) * 2007-01-03 2015-11-03 Monsanto Technology Llc Compositions alimentaires contenant des acides gras a longue chaine supplementaires
US8389808B2 (en) 2007-02-12 2013-03-05 E.I. Du Pont De Nemours And Company Production of arachidonic acid in oilseed plants
ES2559312T3 (es) * 2007-04-03 2016-02-11 E. I. Du Pont De Nemours And Company Multienzimas y su uso en la fabricación de ácidos grasos poliinsaturados
US7794701B2 (en) 2007-04-16 2010-09-14 E.I. Du Pont De Nemours And Company Δ-9 elongases and their use in making polyunsaturated fatty acids
US8765422B2 (en) 2007-07-13 2014-07-01 Dsm Nutritional Products Ag D4 desaturases and D5 elongases
CA2695112A1 (fr) * 2007-07-31 2009-02-05 Basf Plant Science Gmbh Elongases et procedes de production d'acides gras polyinsatures dans des organismes transgeniques
US20090110800A1 (en) * 2007-10-24 2009-04-30 Wilkes Richard S Food compositions incorporating additional long chain fatty acids
WO2009130291A2 (fr) * 2008-04-25 2009-10-29 Basf Plant Science Gmbh Huile végétale de graines
ES2334744B1 (es) * 2008-07-11 2011-04-06 Zurko Research S.L. Procedimiento enzimatico para la obtencion del acido docosahexaenoicoomega-3 y del acido docosapentaenoico omega-6, a partir de aceites vegetales, y sus productos.
US9090902B2 (en) * 2008-08-26 2015-07-28 Basf Plant Science Gmbh Nucleic acids encoding desaturases and modified plant oil
AU2009293160B2 (en) * 2008-09-19 2013-11-21 E. I. Du Pont De Nemours And Company Mutant delta5 desaturases and their use in making polyunsaturated fatty acids
NO2358882T3 (fr) * 2008-11-18 2017-12-23
AU2015224521B2 (en) * 2008-11-18 2017-09-14 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing omega-3 fatty acids
WO2010101220A1 (fr) * 2009-03-04 2010-09-10 国立大学法人九州大学 Nouvelles enzymes et adn codant ces enzymes
EP2430166A1 (fr) 2009-05-13 2012-03-21 BASF Plant Science Company GmbH Acyltransférases et leurs utilisations dans la production d'acide gras
EP2440662B1 (fr) 2009-06-08 2018-08-01 BASF Plant Science Company GmbH Nouveaux composants de l'allongement des acides gras et leur utilisation
US9493520B2 (en) 2009-07-17 2016-11-15 Basf Plant Science Company Gmbh Fatty acid desaturases and elongases and uses thereof
JP2013502914A (ja) 2009-08-31 2013-01-31 ビーエーエスエフ プラント サイエンス カンパニー ゲーエムベーハー 多価不飽和脂肪酸合成の増強を促進する植物の種子特異的遺伝子発現を増強する調節核酸分子
CA3121944A1 (en) 2009-09-24 2011-03-31 Kyushu University, National University Corporation Method for transforming stramenopiles
WO2011064181A1 (fr) 2009-11-24 2011-06-03 Basf Plant Science Company Gmbh Nouvelle désaturase d'acides gras et ses utilisations
US9347049B2 (en) 2009-11-24 2016-05-24 Basf Plant Science Company Gmbh Fatty acid elongase and uses thereof
NO2585603T3 (fr) 2010-06-25 2018-05-19
EP2609203B1 (fr) 2010-08-26 2018-06-20 E. I. du Pont de Nemours and Company Cellules hôtes microbiennes recombinées pour la production élevée d'acide eicosapentaénoïque
AU2011293167B2 (en) 2010-08-26 2016-12-08 E. I. Du Pont De Nemours And Company Mutant delta-9 elongases and their use in making polyunsaturated fatty acids
US9458477B2 (en) 2010-10-21 2016-10-04 Basf Plant Science Company Gmbh Fatty acid desaturases, elongases, elongation components and uses thereof
CN102191257A (zh) * 2011-04-04 2011-09-21 山东农业大学 马铃薯致病疫霉Δ6去饱和酶基因PinD6的克隆及其应用
BR112014025265A2 (pt) 2012-04-12 2017-07-11 Rothamsted Res Ltd produção de ácidos graxos poli-insaturados ômega-3 de cadeia longa
PL2861059T3 (pl) 2012-06-15 2017-10-31 Commw Scient Ind Res Org Wytwarzanie długołańcuchowych wielonienasyconych kwasów tłuszczowych w komórkach roślinnych
CA2879154A1 (fr) 2012-08-03 2014-02-06 Basf Plant Science Company Gmbh Nouvelles enzymes, composants enzymatiques et leurs utilisations
GB201217524D0 (en) * 2012-10-01 2012-11-14 Rothamsted Res Ltd Recombinant organisms
US9309523B2 (en) 2012-12-05 2016-04-12 Exxonmobil Research And Engineering Company Nannochloropsis kozak consensus sequence
CN103173445B (zh) * 2013-04-15 2014-07-30 南京江辉生物科技有限公司 一种欧米伽3的合成基因、含有合成基因的大肠菌及其构建方法
JP2014212732A (ja) * 2013-04-25 2014-11-17 旭硝子株式会社 リシノール酸産生酵母
WO2015001505A2 (fr) 2013-07-05 2015-01-08 Basf Plant Science Company Gmbh Éléments capables de renforcer l'expression ou l'activité génique
NZ721036A (en) 2013-12-18 2023-07-28 Grains Res & Dev Corp Lipid comprising long chain polyunsaturated fatty acids
BR112016014174B1 (pt) 2013-12-18 2022-03-15 BASF Agro B.V. Método de controle de vegetação indesejada e método de produção de uma célula vegetal transgênica
PH12016502586B1 (en) 2014-06-27 2023-07-19 Commw Scient Ind Res Org Lipid comprising docosapentaenoic acid
WO2016075327A2 (fr) 2014-11-14 2016-05-19 Basf Plant Science Company Gmbh Production d'agpi dans des plantes
US10513718B2 (en) 2017-06-06 2019-12-24 City University Of Hong Kong Method of producing polyunsaturated fatty acid
US12241101B2 (en) * 2018-08-10 2025-03-04 Kyowa Hakko Bio Co., Ltd. Microorganism producing polyunsaturated fatty acid and method for producing polyunsaturated fatty acid
CN110373436B (zh) * 2018-12-11 2022-09-23 山东理工大学 一种产双高-γ-亚麻酸卷枝毛霉细胞工厂的构建方法及发酵技术
CA3129494A1 (fr) 2019-02-14 2020-08-20 Cargill, Incorporated Plantes brassica produisant des taux eleves d'acides gras polyinsatures
CN110334869A (zh) * 2019-08-15 2019-10-15 重庆大学 一种基于动态群优化算法的红树林生态健康预测训练方法
AU2021373697A1 (en) 2020-11-04 2023-06-08 Basf Plant Science Company Gmbh Harvest management
EP4312517A1 (fr) 2021-03-25 2024-02-07 BASF Plant Science Company GmbH Gestion d'engrais
JP7852909B2 (ja) * 2022-03-01 2026-04-28 国立大学法人東京海洋大学 タンパク質、遺伝子、ベクター、魚類、不飽和脂肪酸の製造方法、及び魚類の製造方法
WO2023196923A1 (fr) * 2022-04-07 2023-10-12 Checkerspot, Inc. Production d'huile de ricinoléate et ses utilisations
CN116763696B (zh) * 2023-07-31 2025-12-26 广州夕木生物科技有限公司 具有抗炎止痒功效的复合植物提取物及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002090493A2 (fr) † 2001-05-04 2002-11-14 Abbott Laboratories Genes de delta 4-desaturase et leurs applications
US20030163845A1 (en) † 1998-09-02 2003-08-28 Pradip Mukerji Elongase genes and uses thereof
WO2004071467A2 (fr) † 2003-02-12 2004-08-26 E. I. Du Pont De Nemours And Company Production d'acides gras polyinsatures a chaines tres longues dans des plantes a graines oleagineuses
WO2005083093A2 (fr) † 2004-02-27 2005-09-09 Basf Plant Science Gmbh Procede de production d'acides gras polyinsatures chez des plantes transgeniques

Family Cites Families (101)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5352605A (en) 1983-01-17 1994-10-04 Monsanto Company Chimeric genes for transforming plant cells using viral promoters
EP0131623B2 (fr) 1983-01-17 1999-07-28 Monsanto Company Genes chimeriques appropries a l'expression dans des cellules vegetales
US5504200A (en) 1983-04-15 1996-04-02 Mycogen Plant Science, Inc. Plant gene expression
US5420034A (en) 1986-07-31 1995-05-30 Calgene, Inc. Seed-specific transcriptional regulation
DK162399C (da) 1986-01-28 1992-03-23 Danisco Fremgangsmaade til ekspression af gener i baelgplanteceller, dna-fragment, rekombineret dna-fragment samt plasmid til brug ved udoevelsen af fremgangsmaaden
US4962028A (en) 1986-07-09 1990-10-09 Dna Plant Technology Corporation Plant promotors
US5110728A (en) 1986-07-31 1992-05-05 Calgene, Inc. Acyl carrier protein - DNA sequence and synthesis
US5614395A (en) 1988-03-08 1997-03-25 Ciba-Geigy Corporation Chemically regulatable and anti-pathogenic DNA sequences and uses thereof
NZ228320A (en) 1988-03-29 1991-06-25 Du Pont Nucleic acid promoter fragments of the promoter region homologous to the em gene of wheat, dna constructs therefrom and plants thereof
CA1341467C (fr) 1988-07-29 2004-12-07 John C. Rogers Production de polypeptides d'interet commercial a l'aide d'endosperme genetiquement modifie
DE3843628A1 (de) 1988-12-21 1990-07-05 Inst Genbiologische Forschung Wundinduzierbare und kartoffelknollenspezifische transkriptionale regulation
EP0388186A1 (fr) 1989-03-17 1990-09-19 E.I. Du Pont De Nemours And Company Régulation externe de l'expression de gènes
ATE241007T1 (de) 1990-03-16 2003-06-15 Calgene Llc Dnas, die für pflanzliche desaturasen kodieren und deren anwendungen
JPH04506155A (ja) 1990-03-16 1992-10-29 カルジーン，インコーポレイティド 初期種子形成において優先的に発現される新規配列およびそれに関連する方法
US5187267A (en) 1990-06-19 1993-02-16 Calgene, Inc. Plant proteins, promoters, coding sequences and use
WO1993020216A1 (fr) 1991-02-22 1993-10-14 University Technologies International, Inc. Elements cis de proteines des corps oleiferes, servant de signaux de regulation
CA2106960C (fr) 1991-04-09 2005-03-08 Jacqueline De Silva Stimulation de croissance regulant la biosynthese des lipides dans les semences
US5614393A (en) 1991-10-10 1997-03-25 Rhone-Poulenc Agrochimie Production of γ-linolenic acid by a Δ6-desaturase
PH31293A (en) 1991-10-10 1998-07-06 Rhone Poulenc Agrochimie Production of y-linolenic acid by a delta6-desaturage.
CA2124673C (fr) 1991-12-04 2008-08-05 John Browse Genes de desaturase d'acides gras de plantes
CA2084348A1 (fr) 1991-12-31 1993-07-01 David F. Hildebrand Modification de la synthese des acides gras dans des tissus vegetaux transgeniques au moyen d'une d9-desaturase
DE4208050A1 (de) 1992-03-13 1993-09-23 Bayer Ag Azolylmethyl-fluorcyclopropyl-derivate
CA2092069A1 (fr) 1992-03-27 1993-09-28 Asako Iida Plasmide d'expression pour semence
PT637339E (pt) 1992-04-13 2002-03-28 Syngenta Ltd Construcoes de adn e plantas que as incorporam
JPH0662870A (ja) 1992-08-18 1994-03-08 Mitsui Giyousai Shokubutsu Bio Kenkyusho:Kk 大豆ホスホエノールピルビン酸カルボキシラーゼ遺伝子のプロモーター領域及び５’非翻訳領域
DK0668919T3 (da) 1992-11-17 2003-09-15 Du Pont Gener for mikorsomale delta-12-fedtsyredesaturaser og beslægtede enzymer fra planter
JPH08506490A (ja) 1993-02-05 1996-07-16 モンサント・カンパニー 植物中の改変リノレン酸及びリノール酸含量
GB9324707D0 (en) 1993-12-02 1994-01-19 Olsen Odd Arne Promoter
DE733059T1 (de) 1993-12-09 1997-08-28 Univ Jefferson Verbindungen und verfahren zur ortsspezifischen mutation in eukaryotischen zellen
US5576198A (en) 1993-12-14 1996-11-19 Calgene, Inc. Controlled expression of transgenic constructs in plant plastids
BR9408435A (pt) 1993-12-28 1997-08-05 Kirin Brewery Gene para dessaturase de ácido graxo vetor contendo o dito gene planta transformada com o dito gene e processo para criar a dita planta
GB9403512D0 (en) 1994-02-24 1994-04-13 Olsen Odd Arne Promoter
US6310194B1 (en) 1994-09-26 2001-10-30 Carnegie Institution Of Washington Plant fatty acid hydroxylases
GB9421286D0 (en) 1994-10-21 1994-12-07 Danisco Promoter
US5689040A (en) 1995-02-23 1997-11-18 The Regents Of The University Of California Plant promoter sequences useful for gene expression in seeds and seedlings
JP4424756B2 (ja) 1995-07-05 2010-03-03 サッポロビール株式会社 組織特異的プロモーターを用いたトランスジェニック植物およびその作出方法
DE69632403T2 (de) 1995-08-10 2005-05-19 Rutgers University Zellkern-codiertes transkriptionssystem plastiden höherer pflanzen
AU1293297A (en) 1995-12-14 1997-07-03 Cargill Incorporated Plants having mutant sequences that confer altered fatty acid profiles
EP0794250A1 (fr) 1996-03-04 1997-09-10 Soremartec S.A. Isolation et séquence d'un gène FAd2-N du noisetier
DE19626564A1 (de) 1996-07-03 1998-01-08 Hoechst Ag Genetische Transformation von Ciliatenzellen durch Microcarrier-Bombardement mit DNA beladenen Goldpartikeln
US5981841A (en) 1996-08-30 1999-11-09 Monsanto Company Early seed 5' regulatory sequence
US6194167B1 (en) 1997-02-18 2001-02-27 Washington State University Research Foundation ω-3 fatty acid desaturase
US5977436A (en) 1997-04-09 1999-11-02 Rhone Poulenc Agrochimie Oleosin 5' regulatory region for the modification of plant seed lipid composition
AR013633A1 (es) 1997-04-11 2001-01-10 Calgene Llc METODO PARA LA ALTERACIoN DE LA COMPOSICIoN DE ÁCIDOS GRASOS DE CADENA MEDIA EN SEMILLAS VEGETALES QUE EXPRESAN UNA TIOESTERASA QUE PREFIERE CADENA MEDIA VEGETAL HETERoLOGA.
NZ337459A (en) 1997-04-11 2000-07-28 Abbott Lab Nucleic acid construct in plants and dietary supplement
US5968809A (en) 1997-04-11 1999-10-19 Abbot Laboratories Methods and compositions for synthesis of long chain poly-unsaturated fatty acids
US5972664A (en) 1997-04-11 1999-10-26 Abbott Laboratories Methods and compositions for synthesis of long chain poly-unsaturated fatty acids
JP3923144B2 (ja) * 1997-07-31 2007-05-30 大日本印刷株式会社 ホログラム作成方法
JP2001518305A (ja) 1997-09-30 2001-10-16 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 植物種子におけるタンパク質製造
GB9724783D0 (en) 1997-11-24 1998-01-21 Inst Arable Crops Research Novel polypeptides
JP4378452B2 (ja) * 1998-02-09 2009-12-09 豊博 小林 ホログラム構成物
AU747486B2 (en) 1998-03-11 2002-05-16 Novartis Ag Novel plant plastid promoter sequence
CA2330024C (fr) 1998-06-12 2012-01-24 Calgene Llc Acides gras polyinsatures vegetaux
WO1999064616A2 (fr) 1998-06-12 1999-12-16 Abbott Laboratories Acides gras polyinsatures dans des plantes
US6677145B2 (en) 1998-09-02 2004-01-13 Abbott Laboratories Elongase genes and uses thereof
US6403349B1 (en) 1998-09-02 2002-06-11 Abbott Laboratories Elongase gene and uses thereof
US6555732B1 (en) 1998-09-14 2003-04-29 Pioneer Hi-Bred International, Inc. Rac-like genes and methods of use
EP1121150A4 (fr) 1998-10-09 2003-06-04 Merck & Co Inc Desaturase d'acide gras delta 6
CA2791961A1 (fr) 1998-12-07 2000-06-15 Washington State University Research Foundation Desaturases et leurs methodes d'utilisation pour la synthese d'acides gras polyinsatures
WO2000055330A1 (fr) * 1999-03-18 2000-09-21 The University Of Bristol Elongase d'acides gras polyinsatures tires de caenorhabiditis elegans
EE200200443A (et) 2000-02-09 2003-12-15 Basf Aktiengesellschaft Uus elongaasigeen ning polüküllastumata rasvhapete saamise meetod
JP3425622B2 (ja) * 2000-03-30 2003-07-14 独立行政法人産業技術総合研究所 ラビリンチュラ属菌を用いた高度不飽和脂肪酸含有培養物および高度不飽和脂肪酸含有油脂の製造方法
CA2408357A1 (fr) 2000-05-09 2001-11-15 Bioriginal Food & Science Corp. Production d'acides linoleiques et d'acides linoleiques conjugues dans des plantes
CN101845446B (zh) 2000-09-28 2016-01-20 生物源食物及科学公司 脂肪酸去饱和酶家族成员fad4、fad5、fad5-2和fad6及它们的应用
EP1341920A2 (fr) 2000-11-29 2003-09-10 Xenon Genetics, Inc. Genes humains de l'elongase et leurs utilisations
DE10102337A1 (de) 2001-01-19 2002-07-25 Basf Plant Science Gmbh Verfahren zur Herstellung mehrfach ungesättigter Fettsäuren, neue Biosynthesegene sowie neue pflanzliche Expressionskonstrukte
DE10102338A1 (de) 2001-01-19 2002-07-25 Basf Plant Science Gmbh Verfahren zur Expression von Biosynthesegenen in pflanzlichen Samen unter Verwendung von neuen multiplen Expressionskonstrukten
WO2002081668A2 (fr) * 2001-01-25 2002-10-17 Abbott Laboratories Genes desaturase et utilisations de ces derniers
US7238851B2 (en) 2001-03-12 2007-07-03 The General Hospital Corporation Non-human transgenic mammals expressing an n-3 desaturase gene
GB0107510D0 (en) 2001-03-26 2001-05-16 Univ Bristol New elongase gene and a process for the production of -9-polyunsaturated fatty acids
AU2002309856B2 (en) 2001-05-14 2008-10-23 Dsm Ip Assets B.V. Production and use of a polar lipid-rich fraction containing omega-3 and/or omega-6 highly unsaturated fatty acids from microbes, genetically modified plant seeds and marine organisms
US7211656B2 (en) 2002-01-30 2007-05-01 Abbott Laboratories Desaturase genes, enzymes encoded thereby, and uses thereof
DE10219203A1 (de) 2002-04-29 2003-11-13 Basf Plant Science Gmbh Verfahren zur Herstellung mehrfach ungesättigter Fettsäuren in Pflanzen
AU2003258496A1 (en) 2002-07-03 2004-01-23 Basf Plant Science Gmbh Method for the production of conjugated polyunsaturated fatty acids comprising at least two double bonds in plants
CA2510462A1 (fr) 2002-12-19 2004-07-08 University Of Bristol Methode de production d'acides gras polyinsatures dans une plante transgenique ou un micro-organisme comprenant une delta-9-elongase et une delta-8-desaturase introduites
CA2517253C (fr) 2003-02-27 2018-07-03 Basf Plant Science Gmbh Procede de production d'acides gras polyinsatures
EP1613746B1 (fr) 2003-03-31 2013-03-06 University Of Bristol Nouvelles acyltransferases vegetales specifiques pour acides gras a longue chaine polyinsatures
MXPA05010571A (es) 2003-04-08 2005-11-23 Basf Plant Science Gmbh Delta-4-desaturasas a partir de euglena gracilis, que expresan plantas y aceites que comprenden pufa
ES2713548T3 (es) * 2003-08-01 2019-05-22 Basf Plant Science Gmbh Procedimiento para la producción de ácidos grasos poliinsaturados en organismos transgénicos
EP2623584B1 (fr) 2004-02-27 2019-04-10 BASF Plant Science GmbH Procédé de fabrication de plusieurs acides gras insaturés dans des plantes transgéniques
WO2005083053A2 (fr) 2004-02-27 2005-09-09 Basf Plant Science Gmbh Procede pour produire des acides gras $g(v)-3 insatures dans des organismes transgeniques
CA3056110C (fr) 2004-04-22 2020-07-14 Surinder Pal Singh Synthese d'acides gras polyinsatures a chaine longue par des cellules de recombinaison
DE102004060340A1 (de) 2004-07-16 2006-02-09 Basf Plant Science Gmbh Verfahren zur Erhöhung des Gehalts an mehrfach ungesättigten langkettigen Fettsäuren in transgenen Organismen
US7550286B2 (en) 2004-11-04 2009-06-23 E. I. Du Pont De Nemours And Company Docosahexaenoic acid producing strains of Yarrowia lipolytica
DE102005013779A1 (de) 2005-03-22 2006-09-28 Basf Plant Science Gmbh Verfahren zur Herstellung von mehrfach ungesättigten C20- und C22-Fettsäuren mit mindestens vier Doppelbindungen in transgenen Pflanzen
KR200414237Y1 (ko) * 2006-02-06 2006-04-17 주식회사 스토리오브 카메라의 보케 효과용 외부 필터
AR059376A1 (es) 2006-02-21 2008-03-26 Basf Plant Science Gmbh Procedimiento para la produccion de acidos grasos poliinsaturados
WO2009130291A2 (fr) 2008-04-25 2009-10-29 Basf Plant Science Gmbh Huile végétale de graines
EP2281051B1 (fr) 2008-04-30 2015-03-11 Rothamsted Research Limited Désaturase et procédé pour la production d'acides gras polyinsaturés dans des organismes transgéniques
NO2358882T3 (fr) 2008-11-18 2017-12-23
EP2440662B1 (fr) 2009-06-08 2018-08-01 BASF Plant Science Company GmbH Nouveaux composants de l'allongement des acides gras et leur utilisation
US9493520B2 (en) 2009-07-17 2016-11-15 Basf Plant Science Company Gmbh Fatty acid desaturases and elongases and uses thereof
MY181068A (en) 2013-09-25 2020-12-17 Basf Se Recombinant microorganism for improved production of fine chemicals
CN113481217A (zh) 2013-12-13 2021-10-08 巴斯夫欧洲公司 用于精细化学品的改进生产的重组微生物
US10513693B2 (en) 2014-03-19 2019-12-24 Basf Se Use of glycerol with limited feed of carbohydrates for fermentation
PH12016502586B1 (en) 2014-06-27 2023-07-19 Commw Scient Ind Res Org Lipid comprising docosapentaenoic acid
KR20170128509A (ko) 2015-03-18 2017-11-22 바스프 에스이 정제 화학약품의 개선된 제조를 위한 재조합 미생물
EP3303372A2 (fr) 2015-06-04 2018-04-11 Basf Se Micro-organisme recombinant pour la production améliorée de produits chimiques fins
MX386450B (es) 2015-06-12 2025-03-12 Basf Se Microorganismo recombinante para la producción mejorada de productos químicos finos.
JP6502442B2 (ja) * 2017-09-01 2019-04-17 株式会社長英 カメラ用フィルタ
JP2019105703A (ja) * 2017-12-12 2019-06-27 有限会社 高度技術研究所 位相差画像検査装置及び位相差画像検査方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030163845A1 (en) † 1998-09-02 2003-08-28 Pradip Mukerji Elongase genes and uses thereof
WO2002090493A2 (fr) † 2001-05-04 2002-11-14 Abbott Laboratories Genes de delta 4-desaturase et leurs applications
WO2004071467A2 (fr) † 2003-02-12 2004-08-26 E. I. Du Pont De Nemours And Company Production d'acides gras polyinsatures a chaines tres longues dans des plantes a graines oleagineuses
WO2005083093A2 (fr) † 2004-02-27 2005-09-09 Basf Plant Science Gmbh Procede de production d'acides gras polyinsatures chez des plantes transgeniques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABBADI ET AL.: "Transgenic oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty acids?", EUR. J. LIPID. SCI. TECHNOL ., vol. 103, 2001, pages 106 - 113 †
Aktualisierte Liste mit 70 Fettsäuren, die von der allgemeinen Formel l aus Anspruch 8 wie erteilt umfasst sind (eingereicht- mit der Beschwerdebegrundung) †
DREXLER ET AL.: "Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results.", J. PLANT PHYSIOL., vol. 160, no. 7, 2003, pages 779 - 802 †

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